Rapid production of high-value compounds via agrobacterium infiltration of plant tissues

Abstract

The present disclosure provides compositions and methods for Agrobacterium infiltration of plant tissues, for example fruit, vegetable or leaf tissues, and the use of such plant tissues for production of high-value compounds, for example mogroside compounds, in particular mogroside V, betalain, lactoferrin, or fluorescent proteins, such as green fluorescent protein (GFP), and the high-value compounds so produced.

Description

RAPID PRODUCTION OF HIGH-VALUE COMPOUNDS VIA AGROBACTERIUM INFILTRATION OF PLANT TISSUESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Serial No. 63 / 564,641, filed on March 13, 2024, which is herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “ELSS016WO_ST26.xml” which is 618 kilobytes (measured in MS-Windows®) and created on March 12, 2025, and comprises 350 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of genetically engineering plant tissues, and more specifically to methods and compositions for producing plant tissues exhibiting increased production of high-value compounds, for example mogroside compounds, in particular mogroside V, betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP), using Agrobacterium infiltration and transient expression. The present disclosure also relates to the use of such plant tissues to produce recombinant proteins and novel ingredients (e.g., plant extracts, purified and partially purified fractions containing mogrosides) for foods and beverages, and novel foods and beverages (and other compositions of matter) resulting therefrom.BACKGROUND OF THE INVENTION

[0004] Low or non-caloric sweeteners, in particular natural low or non-caloric sweeteners, as an alternative to traditional high calorie sweeteners and artificial sweeteners are becoming increasingly important to the food and beverage industry, in addition to other industries. These alternative sweeteners are used as a substitute for artificial sweeteners or high calorie sweeteners comprising sucrose, fructose, and glucose. Like some artificial sweeteners, some of these alternative sweeteners provide a greater sweetening effect than comparable amounts of caloric sweeteners, and therefore smaller amounts of these alternative sweeteners are required to achieve sweetness comparable to that of sugar. However, some low caloriesweeteners can be expensive to produce and / or possess unfavorable taste characteristics and / or off-tastes, including but not limited to sweetness linger, delayed sweetness onset, negative mouth feel, and bitter, metallic, cooling, astringent, and licorice-like tastes.

[0005] A few natural plants produce low or non-calorie sweeteners. For example, mogrosides, an important class of natural sweeteners, are chemically a class of triterpene glycosides or mogrol glycosides naturally produced by monkfruit (also known as luohan guo; scientific name: Siraitia grosvenorii). Mogrosides contain “zero” calories (less than 5 calories per 8 oz. serving), and are 100-400 times sweeter than sucrose. Mogrosides have also been reported to have a variety of important pharmacological effects. However, although plants like Siraitia grosvenorii make mogrosides, production of mogrosides from these plants is limited and expensive due to the limited natural or agricultural production of these plants. Also, Siraitia grosvenorii prefers to grow in subtropical mountainous regions and requires laborious pollination to set fruits. In addition, production of mogrosides in vitro or in microorganisms has been attempted, but due to extensive processing and other issues has not proven to be economically feasible.

[0006] Therefore there is a need for new compositions and methods for the efficient production of high-value compounds, including, but not limited to, mogrosides.SUMMARY

[0007] The present disclosure solves these and other problems in the art by providing novel compositions and methods for the efficient and cost-effective production of high-value compounds, including, but not limited to, mogrosides and sweet mogrosides, such as those including more than three glucose residues in the molecule, including, but not limited to, mogroside V, isomogroside V, siamenoside I, a-siamenoside I, mogroside IV, mogroside IV A, mogroside III, mogroside III E, mogroside III Al and 11-oxo-mogroside V, or compounds such as betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP), by Agrobacterium infiltration of plant tissues, such as fruits, vegetables or leaves, with a nucleic acid sequence encoding one or more biosynthetic enzymes for production of the high- value compound. The present disclosure also describes the use of this transient plant expression system for the production of recombinant proteins and other molecules of high value.

[0008] The present disclosure provides a method for producing a compound in a fruit, vegetable or leaf tissue, or a portion thereof, comprising a) transforming the fruit, vegetable or leaf tissue, or a portion thereof, by contacting the fruit, vegetable or leaf tissue, or portionthereof, with a suspension of Agrobacterium cells comprising an expression construct comprising at least a first nucleic acid sequence encoding at least a first amino acid sequence capable of synthesizing the compound operably linked to a promoter, b) incubating the transformed fruit, vegetable or leaf tissue, or a portion thereof, for between about 3 and about 14 days, and c) isolating the compound. In certain embodiments, the compound is at least a first mogroside, betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP). In some embodiments, the compound is at least a first mogroside. In other embodiments, the at least a first mogroside is mogroside II A, mogroside II Al , mogroside IT A2, mogroside II E, 11 -oxo-mogroside II, mogroside III, mogroside III Al, mogroside III A2, mogroside III E 11 -oxo-mogroside III, mogroside IV, mogroside IV A, 11 -oxo-mogroside IV, siamenoside I, mogroside V, 11 -oxo-mogroside V or mogroside VI, or an isomer thereof.

[0009] In particular embodiments, the fruit, vegetable or leaf tissue, or a portion thereof, is transformed by injecting, dipping, spraying or pressure delivering to the fruit, vegetable or leaf tissue, or a portion thereof, with the Agrobacterium cells. In further embodiments, the Agrobacterium cells are induced with acetosyringone. In yet further embodiments, the at least a first nucleic acid sequence is transiently expressed.

[0010] In certain embodiments, the fruit, vegetable or leaf tissue is an intact fruit, vegetable or leaf. In some embodiments, the fruit, vegetable or leaf tissue is a fruit, vegetable or leaf that has been sliced. In other embodiments, the fruit, vegetable or leaf tissue is zucchini fruit, cucumber fruit, watermelon fruit, immature watermelon fruit, acorn squash, prickly pear cactus fruit, potato tuber or sugar beet. In yet other embodiments, the fruit, vegetable or leaf tissue, or a portion thereof, is incubated for between about 5 and about 7 days.

[0011] In particular embodiments, the promoter is a heterologous promoter. In further embodiments, the heterologous promoter is an inducible, plant, bacterial, viral, synthetic, constitutive, tissue specific, developmentally regulated, cell cycle regulated, temporally regulated, spatially regulated, and / or spatio-temporally regulated promoter. In still further embodiments, the at least a first nucleic acid sequence is operably linked to a terminator. In yet further embodiments, the terminator is a heterologous terminator. In some embodiments, the nucleic acid sequence further comprises a polynucleotide sequence encoding a screenable marker sequence. In other embodiments, the screenable marker is betalain.

[0012] The present disclosure also provides a composition comprising a compound produced by a method for producing a compound in a fruit, vegetable or leaf tissue, or a portion thereof, comprising a) transforming the fruit, vegetable or leaf tissue, or a portion thereof, by contacting the fruit, vegetable or leaf tissue, or portion thereof, with a suspension ofAgrobacterium cells comprising an expression construct comprising at least a first nucleic acid sequence encoding at least a first amino acid sequence capable of synthesizing the compound operably linked to a promoter, b) incubating the transformed fruit, vegetable or leaf tissue, or a portion thereof, for between about 3 and about 14 days, and c) isolating the compound. In certain embodiments, the compound is at least a first mogroside, betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP). In some embodiments, the compound is at least a first mogroside. In other embodiments, the at least a first mogroside is mogroside II A, mogroside II Al , mogroside IT A2, mogroside II E, 11 -oxo-mogroside II, mogroside III, mogroside III Al, mogroside III A2, mogroside III E 11 -oxo-mogroside III, mogroside IV, mogroside IV A, 11 -oxo-mogroside IV, siamenoside I, mogroside V, 11 -oxo- mogroside V or mogroside VI, or an isomer thereof.

[0013] The present disclosure additionally provides a processed lower calorie food or beverage product produced from a composition comprising a compound produced by a method for producing a compound in a fruit, vegetable or leaf tissue, or a portion thereof, comprising a) transforming the fruit, vegetable or leaf tissue, or a portion thereof, by contacting the fruit, vegetable or leaf tissue, or portion thereof, with a suspension of Agrobacterium cells comprising an expression construct comprising at least a first nucleic acid sequence encoding at least a first amino acid sequence capable of synthesizing the compound operably linked to a promoter, b) incubating the transformed fruit, vegetable or leaf tissue, or a portion thereof, for between about 3 and about 14 days, and c) isolating the compound.

[0014] The present disclosure further provides a method for transient expression of a nucleic acid sequence of interest in a fruit, vegetable or leaf tissue, or a portion thereof, comprising transforming the fruit, vegetable, leaf, or portion thereof, by contacting the fruit, vegetable or leaf tissue, or a portion thereof, with a suspension of Agrobacterium cells comprising an expression construct comprising the nucleic acid sequence of interest, and incubating the transformed fruit, vegetable or leaf tissue, or a portion thereof, for between about 3 and about 14 days.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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 combination with the detailed description of specific embodiments presented herein.

[0016] FIG. 1. A mogroside biosynthetic pathway from Siraitia grosvenorii .

[0017] FIG. 2. Map of expression construct SP3034.

[0018] FIG. 3. Map of expression construct SP3684.

[0019] FIG. 4. Map of expression construct SP5425.

[0020] FIG. 5. Map of expression construct SP5570.

[0021] FIG. 6. Map of expression construct SP5834.

[0022] FIG. 7. Mogroside profile in infiltrated immature watermelon fruit surgical samples.

[0023] FIG. 8. Mogroside profile in infiltrated immature watermelon fruit juice samples.

[0024] FIG. 9. Mogroside profile in infiltrated zucchini fruit surgical samples.

[0025] FIG. 10. Biosynthetic pathway for betalains.

[0026] FIG. 11. Map of expression vector BvCYP76ADl .

[0027] FIG. 12. Map of expression vector BvCYP76AD6.

[0028] FIG. 13. Map of expression vector BvDODAl.

[0029] FIG. 14. Map of expression vector MjcDOPA5GT.

[0030] FIG. 15. Map of expression construct SP5500.

[0031] FIG. 16. Map of expression construct SP6195.

[0032] FIG. 17. Map of expression construct SP5221 .

[0033] FIG. 18. Total mogroside concentration in sunflower cotyledons with the addition of no, IX, 2X or 5X Methyl Jasmonate.

[0034] FIG. 19. Graph of transient mogroside V production of leaf infiltration in lettuce and sugar beet using various expression constructs.

[0035] FIG. 20. Results of promoter testing in lettuce protoplasts.BRIEF DESCRIPTION OF THE SEQUENCES

[0036] SEQ ID NO: 1: cytochrome P450-72 (CYP72 Zm) nucleic acid sequence based on 7.ea mays codon usage.

[0037] SEQ ID NO:2: cytochrome P450-72 (CYP72) amino acid sequence.

[0038] SEQ ID NO:3: cytochrome P450-72 (CYP72 GC) nucleic acid sequence with higher GC content than SEQ ID NO:1.

[0039] SEQ ID NO:4: cucurbitadienol synthase (CDS) nucleic acid sequence.

[0040] SEQ ID NO:5: cucurbitadienol synthase (CDS) amino acid sequence.

[0041] SEQ ID NO:6: cytochrome P450-87 (CYP87) nucleic acid sequence.

[0042] SEQ ID NO:7: cytochrome P450-87 (CYP87) amino acid sequence.

[0043] SEQ ID NO:8: uridine phosphorylase dependent glycosyltransferase-720(UGT720) nucleic acid sequence.

[0044] SEQ ID NO:9: uridine phosphorylase dependent glycosyltransferase-720(UGT720) amino acid sequence.

[0045] SEQ ID NO: 10: uridine phosphorylase dependent glycosyltransferase-94 (UGT94) nucleic acid sequence.

[0046] SEQ ID NO: 11 : uridine phosphorylase dependent glycosyltransferase-94 (UGT94) amino acid sequence.

[0047] SEQ ID NO: 12: squalene epoxidase (SQE) nucleic acid sequence.

[0048] SEQ ID NO: 13: squalene epoxidase (SQE) amino acid sequence.

[0049] SEQ ID NO: 14: epoxy hydrolase (EPH) nucleic acid sequence.

[0050] SEQ ID NO: 15: epoxy hydrolase (EPH) amino acid sequence.

[0051] SEQ ID NO: 16: truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) nucleic acid sequence.

[0052] SEQ ID NO: 17: truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) amino acid sequence.

[0053] SEQ ID NO: 18: NADPH:cytochrome P450 reductase (SgCPR2) nucleic acid sequence.

[0054] SEQ ID NO: 19: NADPH:cytochrome P450 reductase (SgCPR2) amino acid sequence.

[0055] SEQ ID NO:20: uridine phosphorylase dependent glycosyltransferase 74(UGT74_3) nucleic acid sequence.

[0056] SEQ ID NO:21: uridine phosphorylase dependent glycosyltransferase 74(UGT74_3) amino acid sequence.

[0057] SEQ ID NO:22: uridine phosphorylase dependent glycosyltransferase 74(UGT74_4) nucleic acid sequence.

[0058] SEQ ID NO:23: uridine phosphorylase dependent glycosyltransferase 74(UGT74_4) amino acid sequence.

[0059] SEQ ID NO:24: cytochrome P450-87 (C1CYP87D18_B m3) nucleic acid sequence from Citrullus lanatus.

[0060] SEQ ID NO:25: cytochrome P450-87 (C1CYP87D18_B m3) amino acid sequence from Citrullus lanatus.

[0061] SEQ ID NO:26: mutated cytochrome P450-87 (CYP87D17 m2) nucleic acid sequence.

[0062] SEQ ID NO:27: mutated cytochrome P450-87 (CYP87D17 m2) amino acid sequence.

[0063] SEQ ID NO:28: mutated cytochrome P450-87 (CYP87D17 m3) nucleic acid sequence.

[0064] SEQ ID NO:29: mutated cytochrome P450-87 (CYP87D17 m3) amino acid sequence.

[0065] SEQ ID NO:30: mutated cytochrome P450-87 (CYP87D20 m2) nucleic acid sequence.

[0066] SEQ ID NO:31 : mutated cytochrome P450-87 (CYP87D20 m2) amino acid sequence.

[0067] SEQ ID NO:32: mutated cytochrome P450-87 (CYP87D20 m3) nucleic acid sequence.

[0068] SEQ ID NO:33: mutated cytochrome P450-87 (CYP87D20 m3) amino acid sequence.

[0069] SEQ ID NO:34: cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence.

[0070] SEQ ID NO:35: first CYP72 amino acid sequence that can be produced from cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence after cleavage.

[0071] SEQ ID NO:36: second CYP72 amino acid sequence that can be produced from cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence after cleavage.

[0072] SEQ ID NO:37: full length CYP72-2A-CYP72 amino acid sequence from cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence.

[0073] SEQ ID NO:38: uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase dependent glycosyltransferase-720 (UGT720) bicistronic nucleic acid sequence.

[0074] SEQ ID NO:39: first UGT720 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase dependent glycosyltransferase-720 (UGT720) bicistronic nucleic acid sequence after cleavage.

[0075] SEQ ID NO:40: second UGT720 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-uridinephosphorylase dependent glycosyltransferase-720 (UGT720) bicistronic nucleic acid sequence after cleavage.

[0076] SEQ ID NO:41: full length UGT720 amino acid sequence from uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase dependent glycosyltransferase-720 (UGT720) bicistronic nucleic acid sequence.

[0077] SEQ ID NO:42: uridine phosphorylase dependent glycosyltransferase-94 (UGT94)- 2A-uridine phosphorylase dependent glycosyltransferase-94 (UGT94) bicistronic nucleic acid sequence.

[0078] SEQ ID NO:43: first UGT94 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase dependent glycosyltransferase-94 (UGT94) bicistronic nucleic acid sequence after cleavage.

[0079] SEQ ID NO:44: second UGT94 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase dependent glycosyltransferase-94 (UGT94) bicistronic nucleic acid sequence after cleavage.

[0080] SEQ ID NO:45: full length UGT94-2A-UGT94 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase dependent glycosyltransferase-94 (UGT94) bicistronic nucleic acid sequence after cleavage.

[0081] SEQ ID NO:46: truncated 3 -hydroxy-3 -methylglutaryl-CoA reductase (tHMGR)- 2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence.

[0082] SEQ ID NO:47: first tHMGR amino acid sequence that can be produced from truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3- methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence after cleavage.

[0083] SEQ ID NO:48: second tHMGR amino acid sequence that can be produced from truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3- methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence after cleavage.

[0084] SEQ ID NO:49: full length tHMGR-2A-tHMGR amino acid sequence that can be produced from truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence after cleavage.

[0085] SEQ ID NO:50: uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence.

[0086] SEQ ID NO:51: UGT720 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence after cleavage.

[0087] SEQ ID NO:52: CYP72 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence after cleavage.

[0088] SEQ ID NO:53: full length UGT720-2A-CYP72 amino acid sequence from uridine phosphorylase dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) bicistronic nucleic acid sequence.

[0089] SEQ ID NO:54: uridine phosphorylase dependent glycosyltransferase-94 (UGT94)- 2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence.

[0090] SEQ ID NO:55: UGT94 amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3- methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence after cleavage.

[0091] SEQ ID NO:56: tHMGR amino acid sequence that can be produced from uridine phosphorylase dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3- methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence after cleavage.

[0092] SEQ ID NO:57: UGT94-2A-tHMGR amino acid sequence from uridine phosphorylase dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3- methylglutaryl-CoA reductase (tHMGR) bicistronic nucleic acid sequence.

[0093] SEQ ID NO:58: Hygromycin resistance (HygR) gene nucleic acid sequence.

[0094] SEQ ID NO:59: Hygromycin resistance (HygR) gene amino acid sequence.

[0095] SEQ ID NO:60: HLVH12 promoter nucleic acid sequence.

[0096] SEQ ID NO:61 : DCMV promoter nucleic acid sequence.

[0097] SEQ ID NO:62: FMVSgt:PCLSVFlt (also referred to as FSgt / PFLt) chimeric promoter nucleic acid sequence.

[0098] SEQ ID NO:63: Duplicated MMV (dMMV) promoter nucleic acid sequence.

[0099] SEQ ID NO:64: CmYLCV promoter nucleic acid sequence.

[0100] SEQ ID NO:65: CaMV e35S (e35S) promoter nucleic acid sequence.

[0101] SEQ ID NO:66: NOS promoter nucleic acid sequence.

[0102] SEQ ID NO:67: ScBV promoter nucleic acid sequence.

[0103] SEQ ID NO:68: CsVMV promoter nucleic acid sequence.

[0104] SEQ ID NO:69: FMVSgt promoter nucleic acid sequence.

[0105] SEQ ID NO:70 FS1_1 promoter nucleic acid sequence.

[0106] SEQ ID NO:71 FE_3 promoter nucleic acid sequence.

[0107] SEQ ID NO:72 Pea3 A terminator nucleic acid sequence.

[0108] SEQ ID NO:73 At UBQ3 terminator nucleic acid sequence.

[0109] SEQ ID NO:74 Gmax MYB2 terminator nucleic acid sequence.

[0110] SEQ ID NO:75 AIRBCS2B terminator nucleic acid sequence.

[0111] SEQ ID NO:76 Pea E9 terminator nucleic acid sequence.

[0112] SEQ ID NO:77 AtHSPl 8.2 terminator nucleic acid sequence.

[0113] SEQ ID NO:78 Potato Ubi3 terminator nucleic acid sequence.

[0114] SEQ ID NO:79 At Tubulin B9 (AtTub) terminator nucleic acid sequence.

[0115] SEQ ID NO: 80 35S terminator nucleic acid sequence.

[0116] SEQ ID NO: 81 Sy nJ 5UTR nucleic acid sequence.

[0117] SEQ ID NO: 82 TM6 MAR insulator nucleic acid sequence.

[0118] SEQ ID NO: 83 2A self-cleaving peptide nucleic acid sequence.

[0119] SEQ ID NO: 84 2A self-cleaving peptide amino acid sequence.

[0120] SEQ ID NO:85 cytochrome P450-72 (CYP72 V 1) nucleic acid sequence,

[0121] SEQ ID NO: 86 cytochrome P450-72 (CYP72 V 1 ) amino acid sequence,

[0122] SEQ ID NO: 87 alternative squalene epoxidase (SQE) nucleic acid sequence,

[0123] SEQ ID NO:88 alternative cytochrome P450-87 (CYP87) nucleic acid sequence,

[0124] SEQ ID NO: 89 alternative cucurbitadienol synthase (CDS) nucleic acid sequence.

[0125] SEQ ID NQ:90 alternative epoxy hydrolase (EPH) nucleic acid sequence.

[0126] SEQ ID NO:91: alternative uridine phosphorylase dependent glycosyltransferase-720 (UGT720) nucleic acid sequence.

[0127] SEQ ID NO:92: alternative uridine phosphorylase dependent glycosyltransferase- 94 (UGT94) nucleic acid sequence.

[0128] SEQ ID NO:93: upstream terpenoid biosynthetic enzyme (FPS; SP0231), Citrullus lanatus cDNA, nucleic acid sequence.

[0129] SEQ ID NO:94: upstream terpenoid biosynthetic enzyme (FPS; SP0231), Citrullus lanatus, amino acid sequence.

[0130] 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.

[0131] SEQ ID NO:96: cytochrome P450 biosynthetic enzyme, high GC version (CYP87D20 m2 (V2-I46L-A49L-C343Y) GC63; SP0577), Siraitia grosvenorii, nucleic acid sequence.

[0132] SEQ ID NO:97: upstream squalene biosynthetic enzyme (SQS; SP0951), Citrullus lanatus cDNA, nucleic acid sequence.

[0133] SEQ ID NO:98: upstream squalene biosynthetic enzyme (SQS; SP0951), Citrullus lanatus, amino acid sequence.

[0134] SEQ ID NO:99: cytochrome P450 biosynthetic enzyme, mutant, high GC version (CYP87D17 m3 GC63; SP1333), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0135] SEQ ID NO: 100: cytochrome P450 biosynthetic enzyme, mutant, high GC version (CYP87D17 m3 GC63; SP1333), Siraitia grosvenorii, amino acid sequence.

[0136] SEQ ID NO: 101: cytochrome P450 biosynthetic enzyme, mutant, high GC version (CYP87D17 m2 GC63; SP2503), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0137] SEQ ID NO: 102: cytochrome P450 biosynthetic enzyme, mutant high GC version (CYP87D17 m2 GC63; SP2503), Siraitia grosvenorii, protein.

[0138] SEQ ID NO:103: cytochrome p450 reductase biosynthetic enzyme, high GC version (SgCPR2 GC66; SP2571), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0139] SEQ ID NO: 104: cytochrome p450 reductase biosynthetic enzyme, high GC version (SgCPR2 GC66; SP2571), Siraitia grosvenorii, amino acid sequence.

[0140] SEQ ID NO: 105: uridine phosphorylase dependent glycosyltransferase 74, high GC version (SgUGT74_406_2 GC64; SP2666), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0141] SEQ ID NO: 106: uridine phosphorylase dependent glycosyltransferase 74, high GC version (SgUGT74_406_2 GC64; SP2666), Siraitia grosvenorii, amino acid sequence.

[0142] SEQ ID NO: 107: uridine phosphorylase dependent glycosyltransferase 74, high GC version (SgUGT74_345_2 GC65; SP3201), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0143] SEQ ID NO: 108: uridine phosphorylase dependent glycosyltransferase 74, high GC version (SgUGT74_345_2 GC65; SP3201), Siraitia grosvenorii, amino acid sequence.

[0144] SEQ ID NO: 109: cytochrome P450 biosynthetic enzyme, mutant, high GC version (C1CYP87D18_B m3 GC62; SP4900), Citrullus lanatus cDNA, nucleic acid sequence.

[0145] SEQ ID NO: 110: cytochrome P450 biosynthetic enzyme, mutant, high GC version (C1CYP87D18_B m3 GC62; SP4900), Citrullus lanatus, amino acid sequence.

[0146] SEQ ID NO:111: cytochrome P450 biosynthetic enzyme (CYP87D20 m3 (V2- I46L-A49L-C343Y L193KO) GC63; SP4910), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0147] SEQ ID NO: 112: cytochrome P450 biosynthetic enzyme (CYP87D20 m3 (V2- I46L-A49L-C343Y L193KO) GC63; SP4910), Siraitia grosvenorii, amino acid sequence.

[0148] SEQ ID NO: 113: uridine phosphorylase dependent glycosyltransferase(SolycOlg 107825.1 UGT, SP5024), Solanum lycopersicum cDNA, nucleic acid sequence.

[0149] SEQ ID NO: 114: uridine phosphorylase dependent glycosyltransferase(Solyc01gl07825.1 UGT, SP5024), Solanum lycopersicum, amino acid sequence.

[0150] SEQ ID NO: 115: uridine phosphorylase dependent glycosyltransferase(Solyc02g070020.1 UGT; SP5025), Solanum lycopersicum cDNA, nucleic acid sequence.

[0151] SEQ ID NO: 116: uridine phosphorylase dependent glycosyltransferase(Solyc02g070020.1 UGT; SP5025), Solanum lycopersicum, amino acid sequence.

[0152] SEQ ID NO: 117: uridine phosphorylase dependent glycosyltransferase(Solyc09g092500.1 UGT; SP5026), Solanum lycopersicum cDNA, nucleic acid sequence.

[0153] SEQ ID NO: 118: uridine phosphorylase dependent glycosyltransferase(Solyc09g092500.1 UGT; SP5026), Solanum lycopersicum, amino acid sequence.

[0154] SEQ ID NO: 119: uridine phosphorylase dependent glycosyltransferase(Solycl0g085230.2 UGT; SP5027), Solanum lycopersicum cDNA, nucleic acid sequence.

[0155] SEQ ID NO: 120: uridine phosphorylase dependent glycosyltransferase(Solycl0g085230.2 UGT; SP5027), Solanum lycopersicum, amino acid sequence.

[0156] SEQ ID NO: 121: uridine phosphorylase dependent glycosyltransferase(Solycl0g085880.1 UGT; SP5028), Solanum lycopersicum cDNA, nucleic acid sequence.

[0157] SEQ ID NO: 122: uridine phosphorylase dependent glycosyltransferase(Solycl0g085880.1 UGT; SP5028), Solanum lycopersicum, amino acid sequence.

[0158] SEQ ID NO:123: uridine phosphorylase dependent glycosyltransferase 94 (Sg UGT94-289-3 -MSI; SP5034), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0159] SEQ ID NO: 124:- uridine phosphorylase dependent glycosyltransferase 94 (Sg UGT94-289-3 -MSI ; SP5034), Siraitia grosvenorii, amino acid sequence.

[0160] SEQ ID NO:125: upstream terpenoid biosynthetic enzyme (GPS1; SP5035), Cilrullus lanalus cDNA, nucleic acid sequence.

[0161] SEQ ID NO: 126: upstream terpenoid biosynthetic enzyme (GPS 1; SP5035), Citrullus lanatus, amino acid sequence.

[0162] SEQ ID NO:127: uridine phosphorylase dependent glycosyltransferase, high GC version (SgUGT75-281-2 GC65; SP5036), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0163] SEQ ID NO: 128: uridine phosphorylase dependent glycosyltransferase, high GC version (SgUGT75-281-2 GC65; SP5036), Siraitia grosvenorii, amino acid sequence.

[0164] SEQ ID NO: 129: uridine phosphorylase dependent glycosyltransferase 74(SgUGT74ACl (no stop); SP5037), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0165] SEQ ID NO: 130: uridine phosphorylase dependent glycosyltransferase 74(SgUGT74ACl (no stop); SP5037), Siraitia grosvenorii, amino acid sequence.

[0166] SEQ ID NO: 131 : uridine phosphorylase dependent glycosyltransferase (Sg MG1 GC63; SP5038), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0167] SEQ ID NO: 132: uridine phosphorylase dependent glycosyltransferase (Sg MG1 GC63; SP5038), Siraitia grosvenorii, amino acid sequence.

[0168] SEQ ID NO:133: uridine phosphorylase dependent glycosyltransferase (tl34248;SP5039), Stevia rebaudiana cDNA, nucleic acid sequence.

[0169] SEQ ID NO: 134: uridine phosphorylase dependent glycosyltransferase (tl34248;SP5039), Stevia rebaudiana, amino acid sequence.

[0170] SEQ ID NO:135: uridine phosphorylase dependent glycosyltransferase (t72140;SP5040), Stevia rebaudiana cDNA, nucleic acid sequence.

[0171] SEQ ID NO:136:uridine phosphorylase dependent glycosyltransferase (t72140;SP5040), Stevia rebaudiana, amino acid sequence.

[0172] SEQ ID NO:137: uridine phosphorylase dependent glycosyltransferase (172143;SP5041), Stevia rebaudiana cDNA, nucleic acid sequence.

[0173] SEQ ID NO: 138: uridine phosphorylase dependent glycosyltransferase (t72143;SP5041), Stevia rebaudiana, amino acid sequence.

[0174] SEQ ID NO: 139: uridine phosphorylase dependent glycosyltransferase (t74692;SP5042), Stevia rebaudiana cDNA, nucleic acid sequence.

[0175] SEQ ID NO:140: uridine phosphorylase dependent glycosyltransferase (t74692;SP5042), Stevia rebaudiana, amino acid sequence.

[0176] SEQ ID NO: 141 : uridine phosphorylase dependent glycosyltransferase (t85004;SP5043), Stevia rebaudiana cDNA, nucleic acid sequence.

[0177] SEQ ID NO:142: uridine phosphorylase dependent glycosyltransferase (t85004;SP5043), Stevia rebaudiana, amino acid sequence.

[0178] SEQ ID NO: 143: upstream terpenoid biosynthetic enzyme (Sg 000001O9.2_FPS 1;SP5044), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0179] SEQ ID NO: 144: upstream terpenoid biosynthetic enzyme (Sg 00000109.2_FPSl;SP5044), Siraitia grosvenorii, amino acid sequence.

[0180] SEQ ID NO: 145: upstream squalene biosynthetic enzyme (Sg 00000892.729_SQSl; SP5045), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0181] SEQ ID NO: 146: upstream squalene biosynthetic enzyme (Sg00000892.729_SQSl; SP5045), Siraitia grosvenorii, amino acid sequence.

[0182] SEQ ID NO: 147: upstream terpenoid biosynthetic enzyme (Bj HMGS mutant;SP5046), Brassica junea cDNA, nucleic acid sequence.

[0183] SEQ ID NO: 148: upstream terpenoid biosynthetic enzyme (Bj HMGS mutant; SP5046), Brassica junea, amino acid sequence.

[0184] SEQ ID NO: 149: UDPG pyrophosphorylase (Ta UDP-Glucose Pyrophosphorylase;SP5047), Thermocrispum agreste cDNA, nucleic acid sequence.

[0185] SEQ ID NO: 150: UDPG pyrophosphorylase (Ta UDP-Glucose Pyrophosphorylase;SP5047), Thermocrispum agreste, amino acid sequence.

[0186] SEQ ID NO:151: upstream terpenoid biosynthetic enzyme (C1GPS_2; SP5048), Citrullus lanatus cDNA, nucleic acid sequence.

[0187] SEQ ID NO:152: upstream terpenoid biosynthetic enzyme (C1GPS_2; SP5048), Citrullus lanatus, amino acid sequence.

[0188] SEQ ID NO: 153: upstream terpenoid biosynthetic enzyme (MpGPS-SSU; SP5049), Peppermint (Mentha x piperita) cDNA, nucleic acid sequence.

[0189] SEQ ID NO: 154: upstream terpenoid biosynthetic enzyme (MpGPS-SSU; SP5049), Peppermint (Mentha x piperita), amino acid sequence.

[0190] SEQ ID NO: 155: upstream terpenoid biosynthetic enzyme (LcGPS-SSU; SP5050), Litsea cubeba cDNA, nucleic acid sequence.

[0191] SEQ ID NO: 156: upstream terpenoid biosynthetic enzyme (LcGPS-SSU; SP5050), Litsea cubeba, amino acid sequence.

[0192] SEQ ID NO:157: hydrolase biosynthetic enzyme (DbExgl; SP5051), Dekkera bruxellensis cDNA, nucleic acid sequence.

[0193] SEQ ID NO:158: hydrolase biosynthetic enzyme (DbExgl; SP5051), Dekkera bruxellensis, amino acid sequence.

[0194] SEQ ID NO: 159: upstream terpenoid biosynthetic enzyme(C1CGO9GO1156O.1_PMK; SP5072), Citrullus lanatus cDNA, nucleic acid sequence.

[0195] SEQ ID NO: 160: upstream terpenoid biosynthetic enzyme(C1CGO9GO1156O.1_PMK; SP5072), Citrullus lanatus, amino acid sequence.

[0196] SEQ ID NO: 161 : upstream terpenoid biosynthetic enzyme(ClCG10G001230.1_PMK; SP5073), Citrullus lanatus cDNA, nucleic acid sequence.

[0197] SEQ ID NO: 162: upstream terpenoid biosynthetic enzyme(C1CG10G001230. 1_PMK; SP5073), Citrullus lanatus, amino acid sequence.

[0198] SEQ ID NO: 163: upstream terpenoid biosynthetic enzyme(ClCG09G022040.1_IPK; SP5074), Citrullus lanatus cDNA, nucleic acid sequence.

[0199] SEQ ID NO: 164: upstream terpenoid biosynthetic enzyme(ClCG09G022040.1_IPK; SP5074), Citrullus lanatus, amino acid sequence.

[0200] SEQ ID NO: 165: upstream terpenoid biosynthetic enzyme(ClCG05G021710.1_MVD; SP5075), Citrullus lanatus cDNA, nucleic acid sequence.

[0201] SEQ ID NO: 166: upstream terpenoid biosynthetic enzyme(ClCG05G021710.1_MVD; SP5075), Citrullus lanatus, amino acid sequence.

[0202] SEQ ID NO: 167: upstream terpenoid biosynthetic enzyme (Sg00153574. 101_FPSl; SP5076), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0203] SEQ ID NO: 168: upstream terpenoid biosynthetic enzyme (Sg00153574.101_FPSl ; SP5076), Siraitia grosvenorii, amino acid sequence.

[0204] SEQ ID NO:169: upstream terpenoid biosynthetic enzyme (Sg 00010190.2_SQSl;SP5077), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0205] SEQ ID NO: 170: upstream terpenoid biosynthetic enzyme (Sg 00010190.2_SQS 1 ; SP5077), Siraitia grosvenorii, amino acid sequence.

[0206] SEQ ID NO: 171 : upstream terpenoid biosynthetic enzyme (Sg00153449.125_PMK; SP5078), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0207] SEQ ID NO: 172: upstream terpenoid biosynthetic enzyme (Sg00153449. 125_PMK; SP5078), Siraitia grosvenorii, amino acid sequence.

[0208] SEQ ID NO: 173: upstream terpenoid biosynthetic enzyme, high GC version (tHMGR GC69; SP5079), Avena strigose cDNA, nucleic acid sequence.

[0209] SEQ ID NO: 174: upstream terpenoid biosynthetic enzyme, high GC version (tHMGR GC69; SP5079), Avena strigose, amino acid sequence.

[0210] SEQ ID NO: 175: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT720-269-1 Itkin GC65; SP5O8O), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0211] SEQ ID NO: 176: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT720-269-1 Itkin GC65; SP5080), Siraitia grosvenorii, amino acid sequence.

[0212] SEQ ID NO: 177: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT720-269-4 Itkin GC65; SP5O81), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0213] SEQ ID NO: 178: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT720-269-4 Itkin GC65; SP5081), Siraitia grosvenorii, amino acid sequence.

[0214] SEQ ID NO: 179: uridine phosphorylase dependent glycosyltransferase (Sg UGT94-289-2; SP5082), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0215] SEQ ID NO: 180: uridine phosphorylase dependent glycosyltransferase (SgUGT94-289-2; SP5082), Siraitia grosvenorii, amino acid sequence.

[0216] SEQ ID NO: 181 : uridine phosphorylase dependent glycosyltransferase (SgUGT94-289-2 Itkin; SP5083), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0217] SEQ ID NO: 182: uridine phosphorylase dependent glycosyltransferase (SgUGT94-289-2 Itkin; SP5083), Siraitia grosvenorii, amino acid sequence.

[0218] SEQ ID NO: 183: uridine phosphorylase dependent glycosyltransferase (SgUGT94-289-3 Itkin; SP5084), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0219] SEQ ID NO: 184: uridine phosphorylase dependent glycosyltransferase (SgUGT94-289-3 Itkin; SP5084), Siraitia grosvenorii, amino acid sequence.

[0220] SEQ ID NO:185: uridine phosphorylase dependent glycosyltransferase (tl34583;SP5085), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0221] SEQ ID NO:186: uridine phosphorylase dependent glycosyltransferase (tl34583;SP5O85), Siraitia grosvenorii, amino acid sequence.

[0222] SEQ ID NO: 187: uridine phosphorylase dependent glycosyltransferase (174645;SP5086), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0223] SEQ ID NO: 188: uridine phosphorylase dependent glycosyltransferase (t74645;SP5086), Siraitia grosvenorii, amino acid sequence.

[0224] SEQ ID NO: 189: uridine phosphorylase dependent glycosyltransferase (t74693;SP5087), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0225] SEQ ID NO:190: uridine phosphorylase dependent glycosyltransferase (t74693;SP5087), Siraitia grosvenorii, amino acid sequence.

[0226] SEQ ID NO: 191: upstream terpenoid biosynthetic enzyme (Sg 00001291.26_PMK;SP5088), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0227] SEQ ID NO: 192: upstream terpenoid biosynthetic enzyme (Sg 00001291.26_PMK;SP5O88), Siraitia grosvenorii, amino acid sequence.

[0228] SEQ ID NO: 193: upstream terpenoid biosynthetic enzyme (Sg 00154122. 1_IPK;SP5089), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0229] SEQ ID NO:194: upstream terpenoid biosynthetic enzyme (Sg 00154122.1_IPK;SP5089), Siraitia grosvenorii, amino acid sequence.

[0230] SEQ ID NO: 195: upstream terpenoid biosynthetic enzyme (Sg 27366_MVD;SP5090), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0231] SEQ ID NO: 196: upstream terpenoid biosynthetic enzyme (Sg 27366_MVD;SP5090), Siraitia grosvenorii, amino acid sequence.

[0232] SEQ ID NO: 197: hydrolase biosynthetic enzyme (E142A-RRK67; SP5091), Aspergillus oryzae cDNA, nucleic acid sequence.

[0233] SEQ ID NO:198: hydrolase biosynthetic enzyme (E142A-RRK67; SP5091), Aspergillus oryzae, amino acid sequence.

[0234] SEQ ID NO: 199: upstream terpenoid biosynthetic enzyme (Me CDS; SP5092), Momordica charantia cDNA, nucleic acid sequence.

[0235] SEQ ID NO:200: upstream terpenoid biosynthetic enzyme (Me CDS; SP5092), Momordica charantia, amino acid sequence.

[0236] SEQ ID NO:201: upstream terpenoid biosynthetic enzyme (Cp CDS; SP5093), Cucurbita pepo cDNA, nucleic acid sequence.

[0237] SEQ ID NO:202: upstream terpenoid biosynthetic enzyme (Cp CDS; SP5093), Cucurbita pepo, amino acid sequence.

[0238] SEQ ID NO:203: upstream terpenoid biosynthetic enzyme (Tc CDS; SP5094), Trichosanth.es cucumerina cDNA, nucleic acid sequence.

[0239] SEQ ID NO:204: upstream terpenoid biosynthetic enzyme (Tc CDS; SP5094), Trichosanthes cucumerina, amino acid sequence.

[0240] SEQ ID NO:205: upstream terpenoid biosynthetic enzyme (Cc CDS; SP5095), Citrullus colocynthis cDNA, nucleic acid sequence.

[0241] SEQ ID NQ:206: upstream terpenoid biosynthetic enzyme (Cc CDS; SP5095), Citrullus colocynthis, amino acid sequence.

[0242] SEQ ID NO:207: upstream terpenoid biosynthetic enzyme (Ac SQS with intron 6;SP5096), Amaranthus cruentus cDNA, nucleic acid sequence.

[0243] SEQ ID NO:208: upstream terpenoid biosynthetic enzyme (Ac SQS with intron 6;SP5096), Amaranthus cruentus, amino acid sequence.

[0244] SEQ ID NO:209: upstream terpenoid biosynthetic enzyme (Oe SQS; SP5097), Olea europaea cDNA, nucleic acid sequence.

[0245] SEQ ID NO:210: upstream terpenoid biosynthetic enzyme (Oe SQS; SP5097), Olea europaea, amino acid sequence.

[0246] SEQ ID NO:211 : upstream terpenoid biosynthetic enzyme (Cr SQS; SP5098), Chlamydomonas reinhardtii cDNA, nucleic acid sequence.

[0247] SEQ ID NO:212: upstream terpenoid biosynthetic enzyme (Cr SQS; SP5098), Chlamydomonas reinhardtii, amino acid sequence.

[0248] SEQ ID NO:213: epoxidase biosynthetic enzyme (Cp SQE1; SP5099), Cucurbita pepo cDNA, nucleic acid sequence.

[0249] SEQ ID NO:214: epoxidase biosynthetic enzyme (Cp SQE1 ; SP5099), Cucurbita pepo, amino acid sequence.

[0250] SEQ ID NO:215: epoxidase biosynthetic enzyme (Me SQE1; SP5100), Momordica charantia cDNA, nucleic acid sequence.

[0251] SEQ ID NO:2I6: epoxidase biosynthetic enzyme (Me SQE1 ; SP5100), Momordica charantia, amino acid sequence.

[0252] SEQ ID NO:217: epoxidase biosynthetic enzyme (Pg SQE1 ; SP5101), Panax ginseng cDNA, nucleic acid sequence.

[0253] SEQ ID NO:218: epoxidase biosynthetic enzyme (Pg SQE1; SP5101), Panax ginseng, amino acid sequence.

[0254] SEQ ID NO:219: upstream terpenoid biosynthetic enzyme (Ab tHMGR; SP5102), Achyranthes bidentata cDNA, nucleic acid sequence.

[0255] SEQ ID NO:220: upstream terpenoid biosynthetic enzyme (Ab tHMGR; SP5102), Achyranthes bidentata, amino acid sequence.

[0256] SEQ ID NO:221: upstream terpenoid biosynthetic enzyme (Hb tHMGR; SP5103), Hevea brasiliensis cDNA, nucleic acid sequence.

[0257] SEQ ID NO:222: upstream terpenoid biosynthetic enzyme (Hb tHMGR; SP5103), Hevea brasiliensis, amino acid sequence.

[0258] SEQ ID NO:223: upstream terpenoid biosynthetic enzyme (Pg tHMGR; SP5104), Panax ginseng cDNA, nucleic acid sequence.

[0259] SEQ ID NO:224: upstream terpenoid biosynthetic enzyme (Pg tHMGR; SP5104), Panax ginseng, amino acid sequence.

[0260] SEQ ID NO:225: transcription factor (At MYC2D105N mutant; SP5105), Arabidopsis thaliana cDNA, nucleic acid sequence.

[0261] SEQ ID NO:226: transcription factor (At MYC2D105N mutant; SP5105), Arabidopsis thaliana, amino acid sequence.

[0262] SEQ ID NO:227 : transcription Factor (Cs bHLH; SP5106), Cucumis sativus cDNA, nucleic acid sequence.

[0263] SEQ ID NO:228: transcription Factor (Cs bHLH; SP5106), Cucumis sativus, amino acid sequence.

[0264] SEQ ID NO:229: transcription Factor (Bh bHLH; Sp5107), Benincasa hispida cDNA, nucleic acid sequence.

[0265] SEQ ID NO:230: transcription Factor (Bh bHLH; Sp5107), Benincasa hispida, amino acid sequence.

[0266] SEQ ID NO:231 : transcription Factor 2 (Cs bHLH; SP5108), Cucumis sativus cDNA, nucleic acid sequence.

[0267] SEQ ID NO:232: transcription Factor 2 (Cs bHLH; SP51O8), Cucumis sativus, amino acid sequence.

[0268] SEQ ID NO:233: transcription Factor 2 (Bh bHLH; SP5109), Benincasa hispida cDNA, nucleic acid sequence.

[0269] SEQ ID NO:234: transcription Factor 2 (Bh bHLH; SP5109), Benincasa hispida, amino acid sequence.

[0270] SEQ ID NO:235: poplar sucrose synthase (SP5110) Populus alba cDNA, nucleic acid sequence.

[0271] SEQ ID NO:236: poplar sucrose synthase (SP5110) Populus alba, amino acid sequence.

[0272] SEQ ID NO:237 : UDPG pyrophosphorylase (Lg UDP-Glucose Pyrophosphorylase;SP5111), Larix gmelinii cDNA, nucleic acid sequence.

[0273] SEQ ID NO:238: UDPG pyrophosphorylase (Lg UDP-Glucose Pyrophosphorylase;SP5111), Larix gmelinii, amino acid sequence.

[0274] SEQ ID NO:239: transcription factor (At PAP2; SP5112), Arabidopsis thaliana cDNA, nucleic acid sequence.

[0275] SEQ ID NQ:240: transcription factor (At PAP2; SP51 12), Arabidopsis thaliana, amino acid sequence.

[0276] SEQ ID NO:241: heat shock protein (Ms HSP17.6; SP5113), Medicago sativa cDNA, nucleic acid sequence.

[0277] SEQ ID NO:242: heat shock protein (Ms HSP17.6; SP5113), Medicago sativa, amino acid sequence.

[0278] SEQ ID NO:243: transcription factor (At JUNGBRUNNEN1 Nac factor; SP5114), Arabidopsis thaliana cDNA, nucleic acid sequence.

[0279] SEQ ID NO:244: transcription factor (At JUNGBRUNNEN 1 Nac factor; SP5114), Arabidopsis thaliana, amino acid sequence.

[0280] SEQ ID NO:245: upstream terpenoid biosynthetic enzyme (AmGPS-SSU (no stop); SP5115), Antirrhinum majus cDNA, nucleic acid sequence.

[0281] SEQ ID NO:246: upstream terpenoid biosynthetic enzyme (AmGPS-SSU (no stop); SP5115), Antirrhinum majus, amino acid sequence.

[0282] SEQ ID NO:247: upstream terpenoid biosynthetic enzyme (PgFPS; SP5116), Panax ginseng cDNA, nucleic acid sequence.

[0283] SEQ ID NO:248: upstream terpenoid biosynthetic enzyme (PgFPS; SP5116), Panax ginseng, amino acid sequence.

[0284] SEQ ID NO:249: upstream terpenoid biosynthetic enzyme (PgSQS; SP5117), Panax ginseng cDNA, nucleic acid sequence.

[0285] SEQ ID NO:250: upstream terpenoid biosynthetic enzyme (PgSQS; SP5117), Panax ginseng, amino acid sequence.

[0286] SEQ ID NO:251: upstream terpenoid biosynthetic enzyme (gbHMGSl ; SP5118), Ginkgo biloba cDNA, nucleic acid sequence.

[0287] SEQ ID NO:252: upstream terpenoid biosynthetic enzyme (gbHMGSl ; SP5118), Ginkgo biloba, amino acid sequence.

[0288] SEQ ID NO:253: uridine phosphorylase dependent glycosyltransferase(UGT73AM3; SP5263), Cucumis sativus cDNA, nucleic acid sequence.

[0289] SEQ ID NO:254: uridine phosphorylase dependent glycosyltransferase(UGT73AM3; SP5263), Cucumis sativus, amino acid sequence.

[0290] SEQ ID NO:255: uridine phosphorylase dependent glycosyltransferase, high GC version (Solycl0g085230.2 UGT GC61 ; SP5265), Solanum lycopersicum cDNA, nucleic acid sequence.

[0291] SEQ ID NO:256: uridine phosphorylase dependent glycosyltransferase, high GC version (Solycl0g085230.2 UGT GC61 ; SP5265), Solanum lycopersicum, amino acid sequence.

[0292] SEQ ID NO:257: uridine phosphorylase dependent glycosyltransferase, high GC version (UGT73AM3 GC64; SP5350), Cucumis sativus cDNA, nucleic acid sequence.

[0293] SEQ ID NO:258: uridine phosphorylase dependent glycosyltransferase, high GC version (UGT73AM3 GC64; SP5350), Cucumis sativus, amino acid sequence.

[0294] SEQ ID NO:259: AtUBQlO promoter, Arabidopsis thaliana, nucleic acid sequence.

[0295] SEQ ID NO:260: PCLSV promoter, Peanut chlorotic streak virus, nucleic acid sequence.

[0296] SEQ ID NO:261: FS4 promoter, Citrullus lanatus, nucleic acid sequence.

[0297] SEQ ID NO:262: AtACT2 promoter, Arabidopsis thaliana, nucleic acid sequence.

[0298] SEQ ID NO:263: Enhanced AtEf-lA promoter, Cauliflower mosaic virus / Arabidopsis thaliana, nucleic acid sequence.

[0299] SEQ ID NO:264: FuasFScp promoter, Figwort mosaic virus hybrid P, nucleic acid sequence.

[0300] SEQ ID NO:265: AtFAD2 terminator, Arabidopsis thaliana, nucleic acid sequence.

[0301] SEQ ID NO:266: AtNDUFA8 terminator, Arabidopsis thaliana, nucleic acid sequence.

[0302] SEQ ID NO:267: CsHSP17.3 terminator, Cucumis sativus, nucleic acid sequence.

[0303] SEQ ID NO:268: CsHSP22 terminator, Cucumis sativus, nucleic acid sequence.

[0304] SEQ ID NO:269: FE4 promoter, Citrullus lanatus, nucleic acid sequence.

[0305] SEQ ID NO:270: Cucumisin promoter, Cucumis melo L., nucleic acid sequence.

[0306] SEQ ID NO:271: SgCDS promoter, Siraitia grosvenorii, nucleic acid sequence.

[0307] SEQ ID NO:272: upstream terpenoid biosynthetic enzyme, high GC version (CltHMGR GC; SP2040), Citrullus lanatus cDNA, nucleic acid sequence.

[0308] SEQ ID NO:273: upstream terpenoid biosynthetic enzyme, high GC version (CltHMGR GC 2A; SP2040), Citrullus lanatus, amino acid sequenceA

[0309] SEQ ID NO:274: upstream terpenoid biosynthetic enzyme (CltHMGR GC 2A; SP3351), Citrullus lanatus cDNA, nucleic acid sequence.

[0310] SEQ ID NO:275: upstream terpenoid biosynthetic enzyme (CltHMGR GC 2A; SP3351), Citrullus lanatus, amino acid sequence.

[0311] SEQ ID NO:276: upstream terpenoid biosynthetic enzyme (CltHMGR Zm (for 2A);SP4989), Citrullus lanatus cDNA, nucleic acid sequence.

[0312] SEQ ID NO:277 : upstream terpenoid biosynthetic enzyme (CltHMGR Zm (for 2A);SP4989), Citrullus lanatus, amino acid sequence.

[0313] SEQ ID NO:278: cytochrome P450 biosynthetic enzyme, optimized to corn (mf CYP72A459vla Zm (GC48); SP2015), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0314] SEQ ID NO:279: cytochrome P450 biosynthetic enzyme, optimized to com (mf CYP72A459vla Zm (GC48); SP2015), Siraitia grosvenorii, amino acid sequence.

[0315] SEQ ID NO:280: uridine phosphorylase dependent glycosyltransferase (mf UGT720; SP4263), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0316] SEQ ID NO:281: uridine phosphorylase dependent glycosyltransferase (mf UGT720; SP4263), Siraitia grosvenorii, amino acid sequence.

[0317] SEQ ID NO:282: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT94-289-1 GC65; SP4332), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0318] SEQ ID NO:283: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT94-289-1 GC65; SP4332), Siraitia grosvenorii, amino acid sequence.

[0319] SEQ ID NO:284: upstream terpenoid biosynthetic enzyme, optimized for com (Sg CDS Z mays GC51 ; SP5029), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0320] SEQ ID NO:285: upstream terpenoid biosynthetic enzyme, optimized for com (Sg CDS Z mays GC51 ; SP5029), Siraitia grosvenorii, amino acid sequence.

[0321] SEQ ID NO:286: uridine phosphorylase dependent glycosyltransferase, high GC version (mf UGT720 GC; SP5030), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0322] SEQ ID NO:287: uridine phosphorylase dependent glycosyltransferase, high GC version (mf UGT720 GC; SP5030), Siraitia grosvenorii, amino acid sequence.

[0323] SEQ ID NO:288: cytochrome P450 biosynthetic enzyme, high GC version (Sg CYP87D18 GC62; SP5031), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0324] SEQ ID NO:289: cytochrome P450 biosynthetic enzyme, high GC version (Sg CYP87D18 GC62; SP5031), Siraitia grosvenorii, amino acid sequence.

[0325] SEQ ID NO:290: upstream squalene biosynthetic enzyme, high GC version (Sg SQE1 GC66; SP5032), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0326] SEQ ID NO:291: upstream squalene biosynthetic enzyme, high GC version (Sg SQE1 GC66; SP5032), Siraitia grosvenorii, amino acid sequence.

[0327] SEQ ID NO:292: epoxide hydrolase biosynthetic enzyme, high GC version (Sg EPH3 GC64; SP5033), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0328] SEQ ID NO:293: epoxide hydrolase biosynthetic enzyme, high GC version (Sg EPH3 GC64; SP5033), Siraitia grosvenorii, amino acid sequence.

[0329] SEQ ID NO:294: uridine phosphorylase dependent glycosyltransferase, high GC version (SgUGT720 GC 2A; SP3186), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0330] SEQ ID NO:295: uridine phosphorylase dependent glycosyltransferase, high GC version (SgUGT720 GC 2A; SP3186), Siraitia grosvenorii, amino acid sequence.

[0331] SEQ ID NO:296: cytochrome P450 biosynthetic enzyme (Sg SgCYP72vlaZm (for 2A, GC47); SP3204), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0332] SEQ ID NO:297 : cytochrome P450 biosynthetic enzyme (Sg SgCYP72vl a Zm (for 2A, GC47); SP3204), Siraitia grosvenorii, amino acid sequence.

[0333] SEQ ID NO:298: uridine phosphorylase dependent glycosyltransferase (SgUGT94- 1 GC 2A; SP3807), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0334] SEQ ID NO:299: uridine phosphorylase dependent glycosyltransferase (SgUGT94- 1 GC 2A; SP3807), Siraitia grosvenorii, amino acid sequence.

[0335] SEQ ID NO:300: uridine phosphorylase dependent glycosyltransferase (SgUGT720 Zm (for 2A); SP3897), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0336] SEQ ID NO: 301: uridine phosphorylase dependent glycosyltransferase (SgUGT720 Zm (for 2A); SP3897), Siraitia grosvenorii, amino acid sequence.

[0337] SEQ ID NO:302: uridine phosphorylase dependent glycosyltransferase (mf UGT720; SP4263), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0338] SEQ ID NO: 303: uridine phosphorylase dependent glycosyltransferase (mf UGT720; SP4263), Siraitia grosvenorii, amino acid sequence.

[0339] SEQ ID NO:304: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT94-289-1 GC65; SP4332), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0340] SEQ ID NO:305: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT94-289-1 GC65; SP4332), Siraitia grosvenorii, amino acid sequence.

[0341] SEQ ID NO:306: tomato bushy stunt virus pl9 protein (TBSV pl9), nucleic acid sequence.

[0342] SEQ ID NO:307: tomato bushy stunt virus pl9 protein (TBSV pl9), amino acid sequence.

[0343] SEQ ID NO:308: AtNDUFA8 terminator nucleic acid sequence.

[0344] SEQ ID NO:309: AtTUB9 terminator nucleic acid sequence.

[0345] SEQ ID NOG 10: hydroxymethylglutaryl-CoA synthase mutant, nucleic acid sequence.

[0346] SEQ ID NO:311: hydroxymethylglutaryl-CoA synthase mutant, amino acid sequence.

[0347] SEQ ID NOG 12: CsVMV Promoter-SynJ 5' leader, nucleic acid sequence.

[0348] SEQ ID NOG13: RUBY (betalain - dicot), nucleic acid sequence.

[0349] SEQ ID NOG14: RUBY (betalain - dicot), amino acid sequence.

[0350] SEQ ID NOG15: FMV Fit promoter nucleic acid sequence.

[0351] SEQ ID NOG 16: PBI synthetic terminator, nucleic acid sequence.

[0352] SEQ ID NOG 17: cucurbitadienol synthase (SgCDS Zm) nucleic acid sequence from Siraitia grosvenorii, based on Zea mays codon usage, nucleic acid sequence.

[0353] SEQ ID NOG 18: cucurbitadienol synthase (SgCDS Zm) amino acid sequence.

[0354] SEQ ID NOG 19: uridine phosphorylase dependent glycosyltransferase, (Sg UGT720-269-1 ; SP4263), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0355] SEQ ID NOG20: uridine phosphorylase dependent glycosyltransferase, (Sg UGT720-269-1 ; SP4263), Siraitia grosvenorii, amino acid sequence.

[0356] SEQ ID NOG21 : uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT720-269-1 GC; SP5030), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0357] SEQ ID NO:322: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT720-269-1 GC; SP5030), Siraitia grosvenorii, amino acid sequence.

[0358] SEQ ID NO:323: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT94-289-1 ; SP4332), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0359] SEQ ID NO:324: uridine phosphorylase dependent glycosyltransferase, high GC version (Sg UGT94-289-1 ; SP4332), Siraitia grosvenorii, amino acid sequence.

[0360] SEQ ID NO:325: cytochrome P450 biosynthetic enzyme (CYP72vl ; SP1381), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0361] SEQ ID NO:326: cytochrome P450 biosynthetic enzyme (CYP72vl ; SP1381), Siraitia grosvenorii, amino acid sequence.

[0362] SEQ ID NO:327: BvEFl promoter, nucleic acid sequence.

[0363] SEQ ID NO:328: BvGAPDH promoter, nucleic acid sequence.

[0364] SEQ ID NO:329: BvTUB promoter, nucleic acid sequence.

[0365] SEQ ID NO:330:BvUBQ promoter, nucleic acid sequence.

[0366] SEQ ID NO:331: SoUBQ promoter, nucleic acid sequence.

[0367] SEQ ID NO:332: barley alpha-amylase signal peptide, nucleic acid sequence.

[0368] SEQ ID NO:333: barley alpha-amylase signal peptide, amino acid sequence.

[0369] SEQ ID NO:334: mature form of the human lactoferrin gene, nucleic acid sequence.

[0370] SEQ ID NO:335: mature form of the human lactoferrin gene, amino acid sequence.

[0371] SEQ ID NO:336: C-terminal ER retention signal, nucleic acid sequence.

[0372] SEQ ID NO:337: C-terminal ER retention signal, amino acid sequence.

[0373] SEQ ID NO:338: mature form of the bovine lactoferrin gene, nucleic acid sequence.

[0374] SEQ ID NO:339: mature form of the bovine lactoferrin gene, amino acid sequence.

[0375] SEQ ID NO:340: Beta vulgaris (BVMII) promoter, nucleic acid sequence.

[0376] SEQ ID NO:341: Beta vulgaris Cytochrome P450 (CYP76AD1), nucleic acid sequence.

[0377] SEQ ID NO:342: Beta vulgaris cytochrome P450 (CYP76AD1), nucleic acid sequence.

[0378] SEQ ID NO:343: Beta vulgaris cytochrome P450 (CYP76AD6), nucleic acid sequence.

[0379] SEQ ID NO:344: Beta vulgaris cytochrome P450 (CYP76AD6), amino acid sequence.

[0380] SEQ ID NO:345: Beta vulgaris DODA1 (DOPA 4,5 dioxygenase), nucleic acid sequence.

[0381] SEQ ID NO:346: Beta vulgaris DODA1, amino acid sequence.

[0382] SEQ ID NO:347: Mirabilis jalapa cDOPA5GT (cyclo-DOPA5-O-glucosyltransferase), nucleic acid sequence.

[0383] SEQ ID NO:348: Mirabilis jalapa cDOPA5GT, amino acid sequence.

[0384] SEQ ID NO:349: eGFP (green fluorescent protein) with ER retention signal, nucleic acid sequence.

[0385] SEQ ID NO:350: eGFP with ER retention signal, amino acid sequence.DETAILED DESCRIPTION

[0386] The present disclosure generally describes Agrobacterium infiltrated plant tissues for making a variety of high-value compounds, for example mogroside compounds, mogrol / mogroside biosynthetic pathway enzymes, betacyanins and recombinant proteins, betalain, lactoferrin, or fluorescent proteins, such as green fluorescent protein (GFP), and methods for making such transiently expressing plant tissues. The following sections provide embodiments that describe the subject matter in greater detail.I. Mogroside Pathway Enzymes and Mogrosides

[0387] Mogrosides are highly stable molecules based on a triterpene skeleton, formed of varying numbers of glucose units, from 1 to 6 attached to the triterpene backbone. Mogrosides may also comprise a non-glucose moiety, such as grosmomoside I. FIG. 1 shows a mogroside biosynthetic pathway from 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 certain enzymes capable of catalyzing the reactions in the pathway. Importantly, the enzymatic pathway used for production of mogrosides according to the present disclosure is not limited by the mechanisms shown in FIG 1. Other terpene structures, mogrol precursors, enzyme- catalyzed reactions or conversion mechanisms are also possible. Additionally, certain enzymes may catalyze more than one type of reaction, and one or more additional genes can be used to produce precursors, any of the mogrol intermediates, mogrol, or any of the mogroside compounds.

[0388] Provided herein are exemplary nucleic acid and protein sequences for certain mogroside pathway (also referred to as mogroside biosynthetic pathway) enzymes for conversion of mogrol precursors, such as squalene, to mogrol and eventually various mogroside compounds, including, but not limited to, mogroside V. The enzymes of the mogroside pathway include, but are not limited to, squalene epoxidase (SQE), cucurbitadienolsynthase (CDS), epoxy or epoxide hydrolase (EPH), various cytochrome P450 enzymes (CYP), including, but not limited to, CYP72 and CYP87, uridine phosphorylase dependent glycosyltransferase enzymes (UGT), including, but not limited to, UGT720, UGT94 and UGT74, and can additionally include 3 hydroxy-3-methylglutaryl-CoA reductase (HMGR), or truncated versions thereof (tHMGR), and NADPH: cytochrome P450 reductase (CPR2).

[0389] SQE, CDS, CYP and EPH are involved in the successive steps of producing and converting mogrol precursors, such as squalene, into mogrol. Intermediate products of the enzymatic pathway include, but are not limited to, 2,3-oxidosqualene, 2,3;22.23- dioxidosqualene, 24,25 epoxycucurbitadienol, and 24,25 -dihydroxycucurbitadienol. CDS, which is an oxidosqualene cyclase, uses 2,3;22,23-diepoxysqualene as its substrate to produce 24,25-epoxycucurbitadienol. Genomic analysis has revealed that S. grosvenorii has five genes that may encode SQEs. Of these, two are strongly expressed during the initial stages of fruit development, as are CDS, CYP enzymes, and EPHs, which catalyze subsequent steps. The S. grosvenorii genome contains eight genes encoding EPHs, which catalyze conversion of 24,25- epoxycucurbitadienol to 24,25 -dihydroxycucurbitadienol. Certain enzyme may catalyze more than one type of reaction.

[0390] After the formation of mogrol, a series of glycosylations occurs to add glucose molecules, at position C-3 and position C-24, to produce Mogrosides I- VI with various degrees of glycosylation. The Roman numeral I, II, III, IV, V, and VI respectively stand for the number of glucose unit(s) in the corresponding glycosylated mogroside, isomogroside, or oxomogroside. Two UGTs contribute to these steps. One is UGT720, which is strongly expressed in the initial stages of fruit development and transfers one glucose molecule each to the hydroxyl groups at positions C-24 and C 3 of mogrol. The second is UGT94, which is strongly expressed in the latter stages of fruit development and adds sugars to the other sugars already present on the acceptor molecule.

[0391] Although the mogroside pathway was initially described in S. grosvenorii (monkfruit), certain non-monkfruit plants and plant tissues can make tetracyclic triterpenoid compounds similar to mogrol, because at least one of the intermediates, such as triterpenes, exist in the cellular pathway. Moreover, given that the related pathways for modifying tetracyclic triterpenoid compounds require associated enzymes such as reductases, these plants and plant tissues already express certain associated network enzymes. Although such non- monkfruit plants and plant tissues may express enzymes that produce mogrol precursors, these plants and plant tissues do not naturally produce all enzymes in a coordinated fashion required to produce mogrosides. For instance, plants such as cucumber, melon, and watermelonnaturally express cucurbitadienol synthases, which are capable of producing cucurbitadienol. However, other enzymes like the CYP enzymes, which are capable of altering the cucurbitadienol scaffold, redirect this intermediate to other terpene derivatives. Therefore, transient expression of such mogroside pathway genes may allow for non-monkfruit plant tissues to produce mogrol and mogrosides. Therefore, in certain embodiments one or more additional genes could be transiently introduced into non-monkfruit plant tissues, for example fruits or vegetables, to allow for intermediate metabolite production in order to yield mogrol, mogrosides, and mogroside-based sweeteners.

[0392] The term “mogrol precursor” broadly encompasses all possible terpene derivatives and intermediate products towards the production of mogrol and mogroside compounds, including, but not limited to, 2,3-oxidosqualene, 2,3;22.23-dioxidosqualene, 24,25- epoxycucurbitadienol, and 24,25-dihydroxycucurbitadienol, cucurbitadienol, 1 1-hydroxy- cucurbitadienol, and 11 -oxo-cucurbitadienol. Mogrosides refer to any possible glycosylation products of mogrol, including, but not limited to, Siamenoside I, Siratose (a stereoisomer of Siamenoside I), Mogroside VI, Mogroside V, Isomogroside V, Mogroside IV, Mogroside III, Mogroside HIE, Mogroside HE, Mogroside 11A, Mogroside IE, and Mogroside IA. Other examples of mogrosides include, but are not limited to, Mogroside IIB, 7-Oxomogroside IIE, 11 Oxomogroside Al, Mogroside III A2, 11-Deoxymogroside III, 11 -Oxomogroside IVA, 7 Oxomogroside V, and 11-Oxo-mogroside V. Metabolites and derivatives of mogrosides refer to any close variation of mogrosides through a metabolic reaction, naturally occurring reaction, or non-naturally occurring reaction. Derivatives of mogrosides may comprise deletions, alterations, or additions of atom(s) or functional groups compared with standard mogrosides. However, metabolites and derivatives of mogrosides retain substantially the same function and characteristics of standard mogrosides. In certain instances the mogroside compounds produced by the presently disclosed Agrobacterium infiltrated plant tissues can also undergo non-enzymatic (spontaneous) conversion to other mogroside compounds.

[0393] One or more of these mogroside compounds can be isolated, purified or partially purified from the disclosed Agrobacterium infiltrated plant tissues. For example the one or more mogroside compounds can be harvested via wet extraction or an aqueous layer containing the mogroside compounds in bulk processing steps that do not necessarily result in the isolation of a single mogroside compound. Alternatively, Agrobacterium infiltrated plant tissues may be dried and pulverized into powder or extracted, or the Agrobacterium infiltrated plant tissues can be minimally processed and used as a food ingredient.

[0394] In certain embodiments the presently disclosed Agrobacterium infiltrated plant tissues will produce a unique ratio of mogroside compounds. In some embodiments the presently disclosed Agrobacterium infiltrated plant tissues can produce more of one or more of the mogroside compounds and less of other mogroside compounds, for example higher amounts of mogroside V compared to other mogroside compounds.

[0395] In further embodiments the complete mogroside biosynthetic pathway can be established in a selected Agrobacterium infiltrated plant tissue using a combination approach, for example by endogenously activating one or more mogroside biosynthetic pathway nucleic acid sequences that are naturally present in the plant tissue and providing any mogroside biosynthetic pathway nucleic acid sequences that are not naturally present in the plant tissue via one or more expression vector(s) comprising the non-endogenous mogroside biosynthetic pathway nucleic acid sequences.

[0396] In further embodiments the Agrobacterium infiltrated plant tissues can also be engineered to express one or more nucleic acids involved in the biosynthesis of other sweeteners, for example genes involved in the production of siamenoside I, a-siamenoside, steviol glycosides (stevia), Rebaudioside M or glycyrrhizin. Additionally, the Agrobacterium infiltrated plant tissues can be engineered to produce mogrosides and other sweeteners together in the same plant tissue, for example to produce mogrosides and Rebaudioside M.

[0397] In further embodiments the Agrobacterium infiltrated plant tissues can be engineered to express enzymes for the synthesis of other valuable compounds such as betalains or carotenoids, lactoferrin, or fluorescent proteins, such as green fluorescent protein (GFP). Additionally, the Agrobacterium infiltrated plant tissues can be engineered to express recombinant proteins that have research or commercial value.IL Nucleic Acid and Polypeptide Sequences

[0398] Certain embodiments of the current disclosure concern nucleic acid sequences (polynucleotides) and the corresponding amino acid sequences (proteins or polypeptides) for mogroside biosynthesis pathway genes. Complements to any nucleic acid or protein sequences described herein are also provided.

[0399] "Identity," as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. Methods to determine "identity" are designed to give the largest match between thesequences tested. Moreover, methods to determine identity are codified in publicly available programs. "Identity" can be readily calculated by any of the many methods known to those of skill in the art. Computer programs can be used to determine "identity" between two sequences these programs include but are not limited to, GCG; suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, NCBI NLM NIH, Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0400] In accordance with the present disclosure, a polynucleotide or polypeptide sequence as described herein may exhibit at least from about 34%, 40%, 50%, 60%, 62%, 70%, 80%, 85%, 90%, 95%, 99% to about 100% sequence identity to at least one of the sequences set forth herein. For example, in one embodiment, a compound biosynthesis pathway gene as described herein may comprise, for example, 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%, or 100% sequence identity to a sequence selected from SEQ ID NOs:l, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24. 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 85, 87-93, 95, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161 , 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 310, 313, 317, 319, 321, 323, 325, 332, 334, 338, 341, 343, 345, 347 or 349, or a complement thereof. In other embodiments, a compound biosynthesis pathway protein as described herein may comprise for example, 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%, or 100% sequence identity to a sequence selected from 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, 86, 94, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220,222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 311,314, 318, 320, 322, 324, 326, 333, 335, 339, 342, 344, 346, 348 or 350.

[0401] Parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970); Comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, (Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program that can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The above parameters along with no penalty for end gap may serve as default parameters for peptide comparisons.

[0402] Parameters for nucleic acid sequence comparison include the following: Algorithm: Needleman and Wunsch (supra); Comparison matrix: matches=+10; mismatches=0; Gap Penalty: 50; and Gap Length Penalty: 3. A program that can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wis. The above parameters may serve as the default parameters for nucleic acid comparisons.

[0403] As used herein, "hybridization," "hybridizes," or "capable of hybridizing" is understood to mean the forming of a double- or triple-stranded molecule or a molecule with partial double- or triple-stranded nature. Such hybridization may take place under relatively high- stringency conditions, including low salt and / or high temperature conditions, such as provided by a wash in about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C for 10 minutes. In one embodiment of the present disclosure, the conditions are 0.15 M NaCl and 70°C. Stringent conditions tolerate little mismatch between a nucleic acid and a target strand. Such conditions are well-known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like. Also included may be a protein or polypeptide, or fragment thereof, such as any of those set forth herein.

[0404] "Fragment", with respect to the nucleic acid sequences disclosed herein, refers to any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used as probes or primers in hybridization or amplification technologies or in the regulation of replication, transcription or translation. A polynucleotide fragment refers to any subsequence of a polynucleotide,typically, of at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, or at least about 50 nucleotides or more, of any of the nucleic acid sequences provided herein.

[0405] Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide, as disclosed herein. Fragments may have antigenic potential, or may be a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. Fragments can vary in size from as few as 5 amino acids to the full length of the intact polypeptide, but are preferably at least about 10 amino acids in length, at least about 15 amino acids in length, at least about 20 amino acids in length, at least about 25 amino acids in length, at least about 30 amino acids in length, at least about 35 amino acids in length, at least about 40 amino acids in length, at least about 45 amino acids in length, at least about 50 amino acids in length, at least about 55 amino acids in length, or at least about 60 amino acids in length or more, of any of the amino acid sequences provided herein.

[0406] The nucleic acids provided herein as SEQ ID NOs:l, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24. 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 58, 85, 87-93, 95, 96, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 310, 313, 317, 319, 321, 323, 325, 332, 334, 338, 341, 343, 345, 347 or 349 and amino acids provided herein as 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, 86, 94, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204,206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 273, 275, 277, 279, 281,283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 311, 314, 318, 320, 322, 324, 326, 333, 335, 339, 342, 344, 346, 348 or 350, may be from any source, e.g., identified as naturally occurring in a plant, or synthesized, e.g., 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, 87-93, 95, 96, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221 , 223, 225, 227, 229, 231 , 233, 235, 237, 239, 241 , 243, 245, 247, 249, 251, 253, 255, 257, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300,302, 304, 306, 310, 313, 317, 319, 321, 323, 325, 332, 334, 338, 341, 343, 345, 347 or 349, for example to create a coding sequence with a G / C content more like the G / C content of naturally occurring genes from a particular plant. The naturally occurring sequence may be from any plant or algal species, as described herein.III. Transformation Constructs

[0407] Vectors used for creation of Agrobacterium infiltrated plant tissue may include, for example, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning system, as well as fragments of DNA therefrom. Thus when the term “vector” or “expression vector” is used, all of the foregoing types of vectors, as well as nucleic acid sequences isolated therefrom, are included. It is contemplated that utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. In accordance with the present disclosure, this could be used to transiently introduce genes corresponding to an entire biosynthetic pathway into a plant tissue. Introduction of such sequences may be facilitated by use of bacterial or yeast artificial chromosomes (BACs or YACs, respectively), or even plant artificial chromosomes. For example, the use of BACs for Agrobacterium-mediated transformation was disclosed by Hamilton el al. Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996).

[0408] Particularly useful for transient expression are expression cassettes that have been isolated from such vectors. DNA segments used for transient expression in plant tissues will, of course, generally comprise the cDNA, gene or genes that one desires to introduce into and have expressed in the plant tissue. These DNA segments can further include structures such as promoters, enhancers, polylinkers, terminators or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will often encode a protein that will betransiently expressed in the resultant plant tissues resulting in a screenable or selectable trait. The skilled artisan is aware of the genetic elements that must be present on a vector in order to successfully transiently express a sequence of interest in a plant tissue. Components that may be included with vectors used in the current disclosure are as follows.A. Promoters and Other Regulatory Elements

[0409] In certain embodiments, the presently disclosed expression cassettes further comprise one or more promoters, for example one or more of the nucleotide sequences set forth in SEQ ID NOs: 60-71, 259-264, 269-271, 312, 315, 327-331, or 340, or a nucleotide sequence having a sequence identity of 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% to one or more of the nucleotide sequences set forth in SEQ ID NOs:60-71 , 259-264, 269-271 , 312, 315, 327-331 or 340.

[0410] In addition to the promoter sequences disclosed in the Sequence Listing, other exemplary promoters for expression of a nucleic acid sequence include a plant promoter such as the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), or others such as CaMV 19S (Lawton et al., Plant Mol. Biol. 9:315-324, 1987), nos (Ebert et al., Proc. Natl. Acad. Sci. USA 84:5745-5749, 1987), Adh (Walker et al., Proc. Natl. Acad. Sci. USA 84:6624-6628, 1987), sucrose synthase (Yang and Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), a-tubulin, actin (Wang et al., Mol. Cell Biol. 12:3399-3406, 1992), cab (Sullivan et al., Mol. Gen. Genet. 215:431 -440, 1989), PEPCase (Hudspeth and Grula, Plant Mol. Biol. 12:579-589, 1989) or those associated with the R gene complex (Chandler et al., Plant Cell 1:1175-1183, 1989). Tissue specific promoters such as root cell promoters (Conkling et al., Plant Physiol. 93:1203-1211, 1990) and tissue specific enhancers are also contemplated to be useful, as are inducible promoters such as ABA- and turgor-inducible promoters. The PAL2 promoter may in particular be useful with the disclosure (U.S. Patent Application Publication No. 2004 / 0049802, the entire disclosure of which is specifically incorporated herein by reference). In one embodiment of the present disclosure, the native promoter of one or more of the mogroside pathway genes is used. In some embodiments, the promoter is a strong promoter or a weak promoter.

[0411] The DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can also influence gene expression. One may thus wish to employ a particular leader sequence with a transient expression construct of the present disclosure. Leader sequences are contemplated to include those that comprisesequences predicted to direct optimum expression of the attached gene, i.e., to include a consensus leader sequence that may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants may be desirable.

[0412] It is contemplated that vectors for use in accordance with the present disclosure may be constructed to include an ocs enhancer element. This element was first identified as a 16 bp palindromic enhancer from the octopine synthase (ocs) gene of Agrobacterium (Ellis et al., EMBO J. 6:3203-3208, 1987), and is present in at least 10 other promoters (Bouchez et al., EMBO J. 8:4197-4204, 1989). The use of an enhancer element, such as the ocs element and particularly multiple copies of the element, may act to increase the level of transcription from adjacent promoters when applied in the context of transient expression in plant tissues.

[0413] It is envisioned that mogroside biosynthesis pathway coding sequences may be introduced under the control of novel promoters or enhancers, etc., or homologous or tissue specific promoters or control elements. Vectors may include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences. Promoters that direct specific or enhanced transient expression in certain plant tissues will be known to those of skill in the art in light of the present disclosure. These include, for example, the rbcS promoter, specific for green tissue; the ocs, nos and mas promoters that have higher activity in roots or wounded leaf tissue.B. Terminators

[0414] In certain embodiments, the presently disclosed expression cassettes further comprise one or more terminators, for example one or more of the nucleotide sequences set forth in SEQ ID NOs: 72-80, 265-268, 308, 309 or 316, or a nucleotide sequence having a sequence identity of 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% to one or more of the nucleotide sequences set forth in SEQ ID NOs:72-80, 265-268, 308, 309 or 316.

[0415] Transformation constructs for Agrobacterium infiltration of plant tissue prepared in accordance with the present disclosure will typically include a 3' end DNA sequence that acts as a signal to terminate transcription and allow for the polyadenylation of the mRNA produced by coding sequences operably linked to a promoter. In one embodiment of the present disclosure, the native terminator of a mogroside biosynthesis pathway coding sequence is used. Alternatively, a heterologous 3’ end may enhance the expression of sense or antisensemogroside biosynthesis pathway coding sequences. In addition to the terminator sequences disclosed in the Sequence Listing, further examples of terminators that are deemed to be useful in this context include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3’ end) (Bevan et al., Nucl. Acids Res. 11 :369-385, 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato. Regulatory elements such as an Adh intron (Callis et al., Genes Dev. 1:1183-1200, 1987), sucrose synthase intron (Vasil et al., Plant Physiol. 91 : 1575-1579, 1989) orTMV omega element (Gallie and Kado, Proc. Natl. Acad. Sci. USA 86:129-132, 1989), may further be included where desired.C. Transit or Signal Peptides

[0416] In certain embodiments of the present disclosure transit or signal sequences may be incorporated into the mogroside biosynthesis pathway coding sequences. Sequences that are joined to the coding sequence of an expressed gene, which are removed post-translationally from the initial translation product and that facilitate the transport of the protein into or through intracellular or extracellular membranes, are termed transit (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, golgi apparatus and outside of the cellular membrane). By facilitating the transport of the protein into compartments inside and outside the cell, these sequences may increase the accumulation of gene product protecting them from proteolytic degradation. These sequences also allow for additional mRNA sequences from highly expressed genes to be attached to the coding sequence of the genes. Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear in the final polypeptide. It further is contemplated that targeting of certain proteins may be desirable in order to enhance the stability of the protein (U.S. Patent No. 5,545,818, incorporated herein by reference in its entirety).

[0417] Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transformed plant tissue or in directing a protein to the extracellular environment. This generally will be achieved by joining a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particularintracellular, or extracellular destination, respectively, and will then be post-translationally removed.D. Marker Genes

[0418] By employing a screenable marker protein, one can provide or enhance the ability to identify transformants. “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker protein and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode a selectable marker, a trait that one can identify through observation or testing, i.e., by “screening” (e.g., the green fluorescent protein or betalain). In addition to the marker genes disclosed in the Sequence Listing, many additional examples of suitable marker proteins are known to the art and can be employed in the practice of the present disclosure.

[0419] Included within the term “screenable” markers also are genes that encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that are secretable antigens that can be identified by antibody interaction, or even secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g., a- amylase, P-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR S).

[0420] Screenable markers that may be employed include a glucuronidase (GUS) or uidA gene, which encodes an enzyme for which various chromogenic substrates are known; an R- locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a RUBY reporter gene (He et al. Hortic. Res. 7:152, 2020), which encodes enzymes that mediate the production of betalain pigments; a P lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA 75:3737-3741, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80: 1101-1105, 1983), which encodes a catechol dioxygenase that can convert chromogenic catechols; an a- amylase gene (Ikuta et al., Biotechnology 8:241-242, 1990); a tyrosinase gene (Katz et al., I. Gen. Microbiol. 129:2703-2714, 1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form the easily-detectable compound melanin; a P galactosidase gene, which encodes an enzyme for which there arechromogenic substrates; a luciferase (lux) gene (Ow et al., Science 234:856-859, 1986), which allows for bioluminescence detection; an aequorin gene (Prasher et al., Biochem. Biophys. Res. Commun. 126: 1259-1268, 1985), which may be employed in calcium-sensitive bioluminescence detection; or a gene encoding for 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) is also contemplated as a useful reporter gene. Expression of green fluorescent protein may be visualized in a cell or plant as fluorescence following illumination by particular wavelengths of light.E. Suppressors of Silencing

[0421] Suppressors of gene silencing in plants are molecules that interfere with the native RNA silencing pathways, which are crucial for regulating gene expression and defending against viruses. A typical example is the tomato bushy stunt virus 19 protein, which is used to enhance the expression of transgenes in plants by preventing the silencing of these introduced genes (Gao et al. PLoS ONE 8(6): e66046 2013.IV. Antisense and RNAi Constructs

[0422] Antisense and RNAi constructs can be used to knock down the expression of genes encoding enzymes that compete for substrates or intermediates that are used for mogroside production. For example, the native CDS enzyme in zucchini and cucumber that is required to synthesize bitter curcurbitacins can be down-regulated to prevent competition for squalene. This allows the CDS enzyme from monk fruit, which has been shown to mediate the conversion of 2,3;22,23-diepoxysqualene to 24,25-epoxycucurbitadienol (a reaction not commonly associated with other cucurbit CDS enzymes), to favor the production of mogroside endproducts.

[0423] Antisense and RNAi treatments represent one way of altering mogroside biosynthesis pathway gene activity in accordance with the present disclosure (e.g., by down regulation of genes or transcription factors that inhibit expression of mogroside biosynthesis pathway genes or to reduce the levels of enzymes that compete for substrates and intermediates).

[0424] Techniques for RNAi are well known in the art and are described in, for example, Lehner et al., (Brief Funct. Genomic Proteomic 3:68-83, 2004) and Downward (BMJ 328:1245-1248, 2004). The technique is based on the fact that double stranded RNA is capableof directing the degradation of messenger RNA with sequence complementary to one or the other strand (Fire et al., Nature 391 :806-811, 1998). Therefore, by expression of a particular coding sequence in sense and antisense orientation, either as a fragment or longer portion of the corresponding coding sequence, the expression of that coding sequence can be down- regulated.

[0425] Antisense, and in some aspects RNAi, methodology takes advantage of the fact that nucleic acids tend to pair with “complementary” sequences. By complementary, it is meant that polynucleotides are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

[0426] Targeting double- stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and / or stability. Antisense and RNAi constructs, or DNA encoding such RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host plant cell. In certain embodiments of the present disclosure, such an oligonucleotide may comprise any unique portion of a nucleic acid sequence provided herein, or a native gene that needs to be down-regulated. In certain embodiments of the present disclosure, such a sequence comprises at least 18, 20, 25, 30, 50, 75 or 100 or more contiguous nucleic acids of a nucleic acid sequence of interest, and / or complements thereof, which may be in sense and / or antisense orientation. By including sequences in both sense and antisense orientation, increased suppression of the corresponding coding sequence may be achieved.

[0427] Constructs may be designed that are complementary to all or part of the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective constructs may include regions complementary to intron / exon splice junctions. Thus, it is proposed that one embodiment includes a construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether toomuch exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

[0428] As stated above, “complementary” or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences that are completely complementary will he sequences that are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an RNAi or antisense construct that has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see above) could be designed. Methods for selection and design of sequences that generate RNAi are well known in the art (e.g., Reynolds et al., Nat. Biotechnol. 22:326-330, 2004). These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

[0429] It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone may be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence. Constructs useful for generating RNAi may also comprise concatemers of sub-sequences that display gene regulating activity.V. Transformation

[0430] In some embodiments, transiently expressing plant tissues, such as fruits, vegetables or leaves, of the present disclosure are created by transforming the selected natural plants with one or more of the expression cassettes disclosed herein. The natural plants prior to transformation do not naturally produce all mogrol / mogroside pathway enzymes, and do not produce non-native mogrol and mogroside compounds, or produce betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP). Although the natural plants may produce one or more enzymes capable of producing mogrol precursors or mogrol, these plants do not produce non-native mogrosides naturally. In certain embodiments, the selected natural plant tissues for transformation include wild-type, or untransformed, or non-transformed plant tissues, which do not naturally produce detectable amounts of mogrol or mogrosidecompounds, or betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP). In some embodiments, the plant tissue are intact (whole) fruits, vegetables or leaves, while in other embodiments the plant tissues are fruits, vegetables or leaves that have been cut into pieces (for example sliced or diced). Other plants or plant parts that can be transformed with one or more of the expression cassettes disclosed herein include, but are not limited to, zucchini fruit (intact or portions thereof), cucumber fruit (intact or portions thereof), watermelon, immature watermelon fruit (intact or portions thereof), acorn squash (intact or portions thereof), prickly pear cactus fruit (intact or portions thereof), potato tuber (intact or portions thereof, for example sliced), sugar beet (intact or portions thereof, for example sliced), cantaloupe, honeydew, winter melon, casaba melon, Persian melon, citron melon, muskmelon, bailan melon, crenshaw melon, Christmas melon, sprite melon, caravelle melon, hami melon, rocky melon, golden Langkawi melon, Korean melon, saticoy melon, galia melon, jade dew melon, golden prize melon, ten me melon, new century melon, banana melon, yubari king melon, sugar melon, tiger melon, vert grimpant melon, homed melon, cucamelon, casabanana melon, pepino melon, ananas melon, camouflage melon, canary melon, bitter melon, charentais melon, crane melon, SkyRocket melon, honey globe melon, gac melon, autumn sweet melon, snap melon, cucumber, tomato, lettuce, spinach, rice, oat, maize, sorghum, bitter apple (Citrullus colocynthis), pumpkin, chard, tobacco, switch grass, bush cherry, peach, nectarine, apricot, radish, plum, sour cherry, apple, pear, sweet cherry, citrus, zucchini, pea, turnip or Nicotiana benthamiana plants. Mogroside compounds can be isolated from any part of the transformed plant tissue, including, but not limited to, the fruit (juice or rind), leaves, roots, seeds or flowers. The fruits, vegetables or leaves can be fresh from the tree or vine, or fruits, vegetables or leaves that have been transported to a retail outlet for sale.

[0431] Suitable methods for transformation of plant or other cells for use with the current disclosure are believed to include virtually any method by which DNA can be introduced into a cell, such as by Agrobacterium-mediated transformation (U.S. Patent No. 5,591 ,616 and U.S. Patent No. 5,563,055; both specifically incorporated herein by reference in their entirety) and by acceleration of DNA coated particles (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,877; and U.S. Patent No. 5,538,880; each specifically incorporated herein by reference in their entirety), etc. Through the application of techniques such as these, the cells of virtually any plant species may be transiently transformed.A. Agrobacterium -mediated Transformation

[0432] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because the DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. The use of Agrobacterium-mediated plant transformation vectors to introduce DNA into plant cells is well known in the art. See, for example, the methods described by Fraley et al., (Proc. Natl. Acad. Sci. USA 80:4803-4807, 1985), and U.S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety.

[0433] Agrobacterium-mediated transformation is most efficient in dicotyledonous plants and is an efficient method for transformation of dicots, including Arabidopsis, tobacco, tomato, alfalfa and potato. Indeed, while Agrobacterium-mediated transformation has been routinely used with dicotyledonous plants for a number of years, it has only recently become applicable to monocoty ledonous plants. Advances in Agrobacterium-mediated transformation techniques have now made the technique applicable to nearly all monocotyledonous plants. For example, Agrobacterium-mediated transformation techniques have now been applied to rice (Hiei et al. , Plant Mol. Biol. 35:205-218, 1997; U.S. Patent No. 5,591,616, specifically incorporated herein by reference in its entirety), wheat and barley (McCormac et al. , Mol. Biotechnol. 9:155-159, 1998), alfalfa and maize (Ishida et al., Nat. Biotechnol. 14:745-750, 1996). Similarly, Agrobacterium-mediated transformation has also proven to be effective in switchgrass.

[0434] Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations. Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations. In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene transfer.VI. Gene Expression

[0435] While Southern blotting and PCR™ may be used to detect the gene(s) in question, they do not provide information as to whether the corresponding protein is being expressed.Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.

[0436] Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physicalchemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identity of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.

[0437] Assay procedures also may be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and 14C-acetyl CoA or for anthranilate synthase activity by following fluorescence of anthranilate, to name two. In the case of identifying and quantifying mogroside compounds, Liquid Chromatography-Mass Spectrometry (LC-MS) is the preferred method.

[0438] Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including, but not limited to, analyzing changes in the chemical composition, morphology, or physiological properties of the plant. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins that change amino acid composition and may be detected by amino acid analysis, or by enzymes that change starch quantity, which may be analyzed by near infrared reflectance spectrometry.VII. Mogroside Containing Sweeteners and Consumables

[0439] In some embodiments, the present disclosure relates generally to a sweetener or sweetening composition comprising mogroside and / or metabolites or derivatives thereof, wherein the sweetener or sweetening composition is derived from a transiently expressing plant tissues, such as fruits, vegetables or leaves, producing and comprising non-native mogrol / mogrosides. The term "sweetener", as used herein, refers to a consumable product, which produces a sweet taste when consumed alone. In certain embodiments, the sweetener or sweetening composition is derived from the mogrol / mogroside pathway transiently expressing plant tissues, such as fruits, vegetables or leaves, made according to the present disclosure. In some embodiments the sweeteners are high intensity or low intensity sweeteners. Mogroside- containing sweeteners can be derived from the mogrol / mogroside pathway transiently expressing plant or plant tissue, such as fruits, vegetables or leaves, of the present disclosure upon appropriate processing. The resulting sweeteners could be used to provide low or noncaloric sweetness for many purposes. Examples of such uses to provide sweetness are in beverages, such as tea, coffee, fruit juice, and fruit beverages, foods, such as jams and jellies, peanut butter, pies, puddings, cereals, candies, ice creams, yogurts, bakery products; health care products, such as toothpastes, mouthwashes, cough drops, cough syrups; chewing gums; and sugar substitutes.

[0440] In certain embodiments, the sweetener is in a juice of the fruit from a transiently expressing plant tissues, such as fruits, vegetables or leaves, according to the present disclosure. Applications for juice, for example, watermelon juice, containing one or more mogroside compound include, but are not limited to, as a beverage, including, for example, premixed cocktails and dairy alternatives, as an ingredient, for example to be sprayed onto bars or cereal, or used to sweeten ketchup or other common products. In such embodiments the juice can be devitalized, have the protein removed or concentrated.

[0441] In some embodiments, the present disclosure also relates to methods of making a sweetener derived from the presently disclosed transiently expressing plant or plant tissue, such as fruits, vegetables or leaves, producing non-native mogrol / mogrosides. The methods generally encompasses steps that can include, but are not limited to, pre-treatment cleaning and crushing of the transiently expressing plant tissues, such as fruits, vegetables or leaves, or the parts thereof, extraction of the transiently expressing plant or plant tissue, such as fruits, vegetables or leaves, or the parts thereof, sedimentation and / or centrifuge, adsorption and / or separation, concentration and recovery to produce the crude sweetener, further purification,optional concentration / drying, and formulation. Means of extraction encompasses waterextraction at room temperatures, or heated temperature, or refrigerated temperature; extraction via organic solvent such as alcohol, etc. Means of separation and purification encompasses centrifuge, steeping, gravity sedimentation, filtration, micro-filtration, nano filtration, ultrafiltration, reverse osmosis, chromatography, absorption chromatogram, exchanged resin purification, etc.

[0442] In further embodiments the presently disclosed transiently expressing plant tissues, such as fruits, vegetables or leaves, can be processed to produce mogroside-containing ingredients, for example by whole plant extracts, tissue extraction, fruit processing, aqueous separation of small molecules having a mogroside fraction, removal of residual proteins to yield an aqueous fraction free from any genetically engineered components. The resulting mogroside containing ingredient(s) can be in any form, including, but not limited to, a powder, liquid, syrup, concentrate or extract. Additionally in some embodiments a whole mogroside containing fruit or vegetable is the consumable.

[0443] In certain embodiments, the sweetener is obtained from the transiently expressing plant tissues, such as fruits, vegetables, leaves or cotyledons, made according to the present disclosure. In other embodiments, the sweetener is obtained from the fruit, a part of a fruit (e.g., the mesocarp), or other part of an organ or tissue of the transiently expressing plant tissues, such as fruits, vegetables or leaves, made according to the present disclosure.

[0444] Additionally the mogroside compounds produced by the presently disclosed transiently expressing plant tissues, such as fruits, vegetables or leaves, can be blended with one or more other naturally occurring or artificial sweeteners, such as steviol glycosides, siamenoside I, a-siamenoside I, sucrose, glucose, fructose, lactose, maltose, sorbitol, galactose, thaumtin, sucrooctate, bernadame, sucrononic acid, carrelame, lugduname, high fructose corn syrup, RealSweet™ Sugarcane RebM, erythritol, xylitol, yacon syrup, allulose, saccharin, aspartame, acesulfame potassium, sucralose, neotame, advantame, cyclamates or glycyrrhizin. The ratio of the mogroside compound(s) to the other sweetener in the final formulation can be, for example, 10 / 90, 20 / 80. 30 / 70, 40 / 60 / 50 / 50, 60 / 40, 70 / 30, 80 / 20 or 90 / 10, or any other desired ratio. In one embodiment, the ratio is about 80% mogroside V, about 15% 11-oxo- mogroside V and about 5% mogroside III Al. In another embodiment, the ratio is about 40% siamenoside I, about 40% mogroside V and about 20% 11 -oxo-mogroside V.

[0445] In certain embodiments, the one or more additional sweeteners may be a carbohydrate sweetener. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, tagatose,trehalose, galactose, rhamnose, cyclodextrin (e.g., a-cyclodextrin, P-cyclodextrin, and y- cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, xylo-oligosaccharides (xylotriose, xylobiose and the like), gentio- oligoscaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), galactooligosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose (glyceraldehyde), nigero- oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraose, maltotriol, tetrasaccharides, mannan-oligosaccharides, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), dextrins, lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars such as high fructose com / starch syrup (HFCS / HFSS) (e.g., HFCS55, HFCS42, or HFCS90), coupling sugars, soybean oligosaccharides, glucose syrup and combinations thereof. D- or L-configurations can be used when applicable. In other embodiments, the additional sweetener is a carbohydrate sweetener selected from the group consisting of glucose, fructose, sucrose and combinations thereof. In another embodiment, the additional sweetener is a carbohydrate sweetener selected from D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-Arabinose, Turanose and combinations thereof.

[0446] In yet other embodiments, the one or more additional sweeteners is not directly derived from a natural extraction. Such a sweetener characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has less calories. Non-limiting examples of such sweeteners suitable for embodiments of this disclosure include sucralose, potassium acesulfame, acesulfame acid and salts thereof, aspartame, alitame, saccharin and salts thereof, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and salts thereof, neotame, advantame, glucosylated steviol glycosides (GSGs) and combinations thereof. The at least one sweetener not directly derived from natural extraction is present in the sweetener composition in an amount effective to provide a concentration from about 0.3 ppm to about 3,500 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage. In one embodiment, the at least one sweetener not directly derived from natural extraction is present in the sweetener composition in an amount effective to provide a concentration from about 0.5 ppm to about 3,000 ppm, from about 1.0 ppm to about 2,500 ppm, from about 5.0 ppm to about 2,000 ppm, from about 10 ppm to about 1,500 ppm, from about 50 ppm to about 1000 ppm, from about 100 ppm to about 800 ppm, or from about 400 ppm toabout 600 ppm when present in a sweetened beverage. In another embodiment, the at least one embodiment, the at least one sweetener not directly derived from natural extraction is present in the sweetener composition in an amount effective to provide a concentration 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 1000 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage.

[0447] In still other embodiments, the additional sweetener can be a natural high potency sweetener. Suitable natural high potency sweeteners include, but are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, Stevia, stevioside, mogroside IV, mogroside V, Luo Han Guo, miraculin, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hemandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, steviolbioside and cyclocarioside I. The natural high potency sweetener can be provided as a pure compound or, alternatively, as part of an extract. For example, rebaudioside A can be provided as a sole compound or as part of a Stevia extract. The natural high potency sweetener is present in the sweetener composition in an amount effective to provide a concentration from about 0.1 ppm to about 3,000 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage. In one embodiment, the natural high potency sweetener is present in the sweetener composition in an amount effective to provide a concentration from about 0.5 ppm to about 2500 ppm, from about 1.0 ppm to about 2000 ppm, from about 5 ppm to about 1500 ppm, from about 10 ppm to about 1000 ppm, or about 25 ppm to about 500 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage. In one embodiment, the natural high potency sweetener is present in the sweetener composition in an amount effective to provide a concentration 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 300 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage.

[0448] In still other embodiments, the additional sweetener can be chemically or enzymatically modified natural high potency sweetener. Modified natural high potency sweeteners include glycosylated natural high potency sweetener such as glucosyl-, galactosyl- , or fructosyl-derivatives containing 1-50 glycosidic residues. Glycosylated natural high potency sweeteners may be prepared by an enzymatic transglycosylation reaction catalyzed by various enzymes possessing transglycosylating activity.

[0449] When the sweetener composition contains more than one sweetener, the sweeteners may exhibit synergy when combined and have improved flavor and temporal profiles compared to each sweetener alone. As used herein, the term "temporal profile" of a composition means the intensity of sweetness perceived over time in tasting of a composition by a human. The term "flavor profile" or "taste profile," as generally used herein, refers to the intensity of various flavor / taste attributes of a sweetener or sweetened composition. Exemplary flavor / taste attributes are sweetness intensity, bitterness intensity, salty intensity, licorice intensity, cooling intensity, and licorice intensity. Methods of determining the flavor profile of a given sweetener or sweetened composition are known in the art. The term "synergistic" or "synergistic effect" refers to an effect (e.g., flavor, temporal profile) achieved with the combination of two or more sweeteners which is greater than the sum of the effects that effect from using the particular sweeteners alone or separately. Advantageously, such synergy between the two or more sweeteners allows for the use of smaller doses of one or both sweeteners or provides greater effect at the same amounts. The amount or degree of synergism may vary.

[0450] The amount of sucrose in a reference solution may be described in degrees Brix (°Bx). One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by weight (% w / w) (strictly speaking, by mass). In one embodiment, a sweetener composition contains one or more of the presently disclosed sweetener compounds in an amount effective to provide sweetness equivalent from of at least about 5 degrees Brix of sugar when present in a sweetened composition, such as, for example, from at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11 , at least about 12, at least about 13, at least about 14 or at least about 15 or more degrees Brix.

[0451] The sweetness of a non-sucrose sweetener can also be measured against a sucrose reference by determining the non-sucrose sweetener's sucrose equivalence. Typically, taste panelists are trained to detect sweetness of reference sucrose solutions containing between 1- 15% sucrose (w / v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percentsucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then the sweetener is said to be 10 times as potent as sucrose.

[0452] The sweetener compositions can be customized to provide the desired calorie content. For example, sweetener compositions can be "full-calorie", such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, a food, other consumable or beverage) and have about 120 calories per 8 oz. serving. Alternatively, sweetener compositions can be "mid-calorie", such that they impart the desired sweetness when added to a sweetenable composition and have less than about 60 calories per 8 oz. serving. In other embodiments, sweetener compositions can be "low-calorie", such that they impart the desired sweetness when added to a sweetenable composition and have less than 40 calories per 8 oz. serving. In still other embodiments, the sweetener compositions can be "zero-calorie", such that they impart the desired sweetness when added to a sweetenable composition and have less than 5 calories per 8 oz. serving.

[0453] The presently disclosed the sweetener compositions can optionally include one or more additional additives. In some embodiments, the sweetener composition contains additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighing agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers and combinations thereof. In some embodiments, the additives act to improve the temporal and flavor profile of the sweetener to provide a sweetener composition with a taste similar to sucrose. The sweetened compositions can contain one or more functional ingredients, as detailed above. Functional ingredients include, but are not limited to, vitamins, minerals, antioxidants, preservatives, glucosamine, polyphenols and combinations thereof. Any suitable functional ingredient described herein can be used.

[0454] There is a beneficial environmental impact of the presently disclosed mogroside sweeteners compared to existing monkfruit production. The presently disclosed mogroside sweeteners enables local production, resulting in less transportation and fewer food miles as a result, compared to for example harvesting and processing only in China and shipping across the globe to food companies. The presently disclosed mogroside sweeteners also require minimal processing because of the ease of access to the mogroside sweeteners in the presentlydisclosed transliently expressing plants tissues compared to, for example, factory processing of monkfruit sweetener in China.VIII. Sweetened Compositions

[0455] The presently disclosed sweetener compositions can be incorporated in any known edible material (referred to herein as a "sweetenable composition"), such as, for example, pharmaceutical compositions, edible gel mixes and compositions, dental compositions, foodstuffs (confections, condiments, chewing gum, cereal compositions baked goods dairy products, and tabletop sweetener compositions) beverages and beverage products.

[0456] The sweetened compositions disclosed here include beverages, i.e., ready to drink liquid formulations, beverage concentrates and the like. In certain embodiments, beverage concentrates are prepared with an initial volume of liquid (e.g., water) to which the additional ingredients are added. Full strength beverage compositions can be formed from the beverage concentrate by adding further volumes of liquid (e.g., water) to the concentrate.

[0457] In embodiments of sweetenable compositions using mogroside-containing filler juice concentrate (about 80% mogroside V, about 15% 1 1-oxo-mogroside V and about 5% mogroside III Al), studies have shown the taste to be the sweetest and cleanest tasting natural sweetener. The presently disclosed mogroside-containing filler juice concentrate can be produced from consumer- friendly fruits that can be locally grown with sustainable production, is the only sweetener for high sugar reduction while maintaining 100% juice labeling, and is an affordable drop in solution. The filler juice may be used as single strength or concentrated to deliver clean, sweet taste across various inclusion levels. The filler juice mogroside concentrations can deliver the equivalent of ~10 sucrose equivalent value (SEV) when used at various formula inclusions.

[0458] Besides concentration, other juice parameters are also readily changeable, resulting in different sweetener products. For example, the natural sugars in the fruit (for example watermelon) may be partially or fully removed, the juice color and / or flavor may be minimized or removed, the pulp may be removed as is typical, or remain fully or partially as in purees, and the acidity may be reduced, or combinations of one or more of these parameters can be changed.

[0459] Filler juice applications include, but are not limited to, juices, nectars, fruit / flavored still drinks, energy and sports drinks, carbonated soft drinks, flavored waters, nutritional drinks, vitamins and dietary supplements or oral rehydration in the form of liquids or chews / gummies, snacks such as snack bars or fruit snacks, sugar and gum confectionary in the form of jelliesand chews, dairy products such as spoonable yogurt, drinking yogurt and flavored drinks, desserts, ice cream, frozen yogurt, water-based ice pops and sorbets, breakfast cereals and other cold cereals, tabletop sweeteners, sweet spreads such as syrups and fruit spreads, sauces and seasonings such as table sauces and cooking sauces, and processed and packaged fruit and vegetables.

[0460] In embodiments of sweetenable compositions using mogroside-containing dry powder (about 40% siamenoside I, about 40% mogroside V, and about 20% 11-oxo- mogroside V), studies have shown the taste to be clean, with high levels of sweetness with no off-tastes in demanding applications. The presently disclosed mogroside-containing dry powder can be produced from consumer-friendly vegetables that can be locally grown with sustainable production, results in sugar and calorie reduction with strong positive associations to health benefits, and is a fraction of monk fruit / parity with sucrose. The dry powder may be used across a wide range of food and drink applications to deliver the cleanest, sweetest taste at low inclusion levels. The dry powder concentrations can deliver the equivalent of ~10 SEV when used at various purity levels.

[0461] Dry powder application include, but are not limited to: wellness and functional drinks, such as energy and sports drinks, carbonated soft drinks, flavored waters, juices, nectars, fruit / flavored still drinks, protein and meal replacement drinks, drink mixes, drink concentrates, ready-to-drink tea and ready-to-drink coffee; dietary supplements and over-the- counter products, such as vitamins and dietary supplements, oral hydration, cold relief, digestive treatments, sleep aids, pain relief in capsule, tablet, liquid, powder, chew / gummy, lozenge and other formats; snacks such as snack bars, fruit snacks, nuts, trail mixes, com rice, potato and wheat snacks; bakery products such as cookies, cakes and sweet goods, baking mixes and ingredients and breads; dairy and desserts such as spoonable and drinking yogurt, flavored drinks, creamers, ice cream and frozen yogurt, water-based ice pops and sorbets, shelfstable desserts and dessert toppings; hot and cold breakfast cereals; artificial and other natural sweeteners (tabletop sweeteners); sugar and chocolate confectionary such as jellies and chews, mints, gum, toffee and caramels, marshmallows and various chocolate formats; sweet spreads such as syrups, fruit, nut and chocolate spreads; sauces and seasonings such as table, cooking and pasta sauces, vinegar and dressings and pickled condiments; meals and processed meats such as prepared meals, meal kits, sandwiches and wraps and poultry and meat products; and processed and packaged fruit and vegetables.A. Beverage and Beverage Products

[0462] In one embodiment, the sweetened composition is a beverage or beverage product. "Beverage product", as used herein, is a ready-to-drink beverage, a beverage concentrate, a beverage syrup, or a powdered beverage. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Carbonated beverages include, but are not limited to, frozen carbonated beverages, enhanced sparkling beverages, cola, fruit-flavored sparkling beverages (e.g., lemon- lime, orange, grape, strawberry and pineapple), ginger-ale, soft drinks and root beer. Non-carbonated beverages include, but are not limited to, fruit juice, fruit-flavored juice or water, juice drinks, nectars, fruit / flavored still drinks, energy and sports drinks, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, nutritional drinks, enhanced water drinks, enhanced water with vitamins, near water drinks (e.g., water with natural or synthetic flavorants), coconut water, tea type drinks (e.g., black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g., milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages), beverages containing cereal extracts and smoothies.

[0463] In certain embodiments, the beverage is a juice beverage that has been modified to remove at least some sucrose. In certain embodiments, such juice may be modified to remove at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more of the sucrose in the non-modified juice. In certain embodiments, the modification occurs through filtration of such juice to remove sucrose. In certain embodiments, sucrose in the juice is broken down to fructose and glucose, prior to adding the sweetening composition described herein.

[0464] Beverages comprise a matrix, i.e., the basic ingredient in which the ingredients- including the compositions of the present disclosure— are dissolved. In one embodiment, a beverage comprises water of beverage quality as the matrix, such as, for example deionized water, distilled water, reverse osmosis water, carbon-treated water, purified water, demineralized water and combinations thereof, can be used. Additional suitable matrices include, but are not limited to phosphoric acid, phosphate buffer, citric acid, citrate buffer and carbon-treated water. Beverage concentrates and beverage syrups are prepared with an initial volume of liquid matrix (e.g., water) and the desired beverage ingredients. Full strength beverages are then prepared by adding further volumes of water. Powdered beverages are prepared by dry-mixing all of the beverage ingredients in the absence of a liquid matrix. Full strength beverages are then prepared by adding the full volume of water.

[0465] It is contemplated that the pH of the beverage does not materially or adversely affect the taste of the sweetener. A non-limiting example of the pH range of the beverage may be from about 1 .8 to about 10. In one embodiment, the pH of the beverage is about 4. In another embodiment, the pH of the beverage is less than about 4. In a particular embodiment, the pH of the beverage 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 about 2.2. In another embodiment, the pH of the beverage is about 3.8, about 3.6, about 3.4, about 3.2, about 3.0, about 2.8, about 2.6, about 2.4 or about 2.2 or less.B. Edible Gel Mixes and Edible Gel Compositions

[0466] In one embodiment, the sweetened composition is an edible gel or edible gel mix. Edible gels are gels that can be eaten. Non-limiting examples of edible gel compositions for use in particular embodiments include gel desserts, puddings, jellies, pastes, trifles, aspics, marshmallows, gummy candies / chews, or the like. Edible gel mixes generally are powdered or granular solids to which a fluid may be added to form an edible gel composition. Nonlimiting examples of fluids for use in particular embodiments include water, dairy fluids, dairy analogue fluids, juices, alcohol, alcoholic beverages, and combinations thereof. Non-limiting examples of dairy fluids which may be used in particular embodiments include milk, cultured milk, cream, fluid whey, and mixtures thereof. Non-limiting examples of dairy analogue fluids which may be used in particular embodiments include, for example, soy milk and non-dairy coffee whitener.C. Confections

[0467] In one embodiment, the sweetened composition is a confection. As referred to herein, "confection" can mean a sweet, a loll ie, a confectionery, or similar term. The confection generally contains a base composition component and a sweetener component. According to particular embodiments of the present disclosure, the confections may be desserts such as yogurt, jellies, drinkable jellies, puddings, Bavarian cream, blancmange, cakes, brownies, mousse and the like, sweetened food products eaten at tea time or following meals; frozen foods; cold confections, e.g., types of ice cream such as ice cream, ice milk, lacto-ice and the like, and ice confections such as sherbets, dessert ices and the like; general confections, e.g., baked confections or steamed confections such as crackers, biscuits, buns with bean-jam filling, halvah, alfajor, and the like; rice cakes and snacks; table top products; general sugar confections such as chewing gum, hard candy, soft candy, mints, nougat candy, jelly beans,fudge, toffee, taffy, Swiss milk tablet, licorice candy, chocolates, gelatin candies, marshmallow, marzipan, divinity, cotton candy, and the like; sauces including fruit flavored sauces, chocolate sauces and the like; edible gels; cremes including butter cremes, flour pastes, whipped cream and the like; jams including strawberry jam, marmalade and the like; and breads including sweet breads and the like or other starch products, and combinations thereof.D. Condiment Compositions

[0468] In one embodiment, the sweetened composition is a condiment composition. Condiments, as used herein, are compositions used to enhance or improve 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; jellies, jams, marmalades, or preserves; mayonnaise; peanut butter; relish; remoulade; salad dressings, salsa; sauerkraut; soy sauce; steak sauce; syrups; tartar sauce; and Worcestershire sauce. Condiment bases generally comprise a mixture of different ingredients, non-limiting examples of which include vehicles (e.g., water and vinegar); spices or seasonings (e.g., salt, pepper, garlic, mustard seed, onion, paprika, turmeric, and combinations thereof); fruits, vegetables, or their products (e.g., tomatoes or tomato-based products (paste, puree), fruit juices, fruit juice peels, and combinations thereof); oils or oil emulsions, particularly vegetable oils; thickeners (e.g., xanthan gum, food starch, other hydrocolloids, and combinations thereof); and emulsifying agents (e.g., egg yolk solids, protein, gum arabic, carob bean gum, guar gum, gum karaya, gum tragacanth, carrageenan, pectin, propylene glycol esters of alginic acid, sodium carboxymethyl-cellulose, polysorbates, and combinations thereof). Recipes for condiment bases and methods of making condiment bases are well known to those of ordinary skill in the art.E. Chewing Gum Compositions

[0469] In one embodiment, the sweetened composition is a chewing gum composition. Chewing gum compositions generally comprise a water-soluble portion and a water-insoluble chewable gum base portion. The water soluble portion dissipates with a portion of the flavoring agent over a period of time during chewing while the insoluble gum base portion is retained in the mouth. The insoluble gum base generally determines whether a gum is considered chewing gum, bubble gum, or a functional gum.

[0470] Flavoring agents may be used in either the insoluble gum base or soluble portion of the chewing gum composition. Such flavoring agents may be natural or artificial flavors. In aparticular embodiment, the flavoring agent comprises an essential oil, such as an oil derived from a plant or a fruit, peppermint oil, spearmint oil, other mint oils, clove oil, cinnamon oil, oil of wintergreen, bay, thyme, cedar leaf, nutmeg, allspice, sage, mace, and almonds. In another particular embodiment, the flavoring agent comprises a plant extract or a fruit essence such as apple, banana, watermelon, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, and mixtures thereof. In still another particular embodiment, the flavoring agent comprises a citrus flavor, such as an extract, essence, or oil of lemon, lime, orange, tangerine, grapefruit, citron, or kumquat.F. Cereal Compositions

[0471] In one embodiment, the sweetened composition is a cereal composition. Cereal compositions typically are eaten either as staple foods or as snacks. Non-limiting examples of cereal compositions for use in particular embodiments include ready-to-eat cereals as well as hot cereals. Ready-to-eat cereals are cereals which may be eaten without further processing (i.e., cooking) by the consumer. Examples of ready-to-eat cereals include breakfast cereals and snack bars. Breakfast cereals typically are processed to produce a shredded, flaky, puffy, or extruded form. Breakfast cereals generally are eaten cold and are often mixed with milk and / or fruit. Snack bars include, for example, energy bars, rice cakes, granola bars, and nutritional bars. Hot cereals generally are cooked, usually in either milk or water, before being eaten. Non-limiting examples of hot cereals include grits, porridge, polenta, rice, and rolled oats.

[0472] Cereal compositions generally comprise at least one cereal ingredient. As used herein, the term "cereal ingredient" denotes materials such as whole or part grains, whole or part seeds, and whole or part grass. Non-limiting examples of cereal ingredients for use in particular embodiments include maize, wheat, rice, barley, bran, bran endosperm, bulgur, sorghums, millets, oats, rye, triticale, buckwheat, fonio, quinoa, bean, soybean, amaranth, teff, spelt, and kaniwa.G. Baked Goods

[0473] In one embodiment, the sweetened composition is a baked good. "Baked goods," as used herein, include ready to eat and all ready to bake products, flours, and mixes requiring preparation before serving. Non-limiting examples of baked goods include cakes, crackers, cookies, brownies, muffins, rolls, bagels, donuts, strudels, pastries, croissants, biscuits, bread, bread products, and buns.

[0474] Baked goods in accordance with particular embodiments of this disclosure generally comprise a combination of sweetener, water, fat and leavening agent. Baked goods made in accordance with many embodiments of this disclosure also contain flour in order to make a dough or a batter.

[0475] According to particular embodiments of this disclosure, leavening agents may comprise chemical leavening agents or yeast leavening agents. Non-limiting examples of chemical leavening agents suitable for use in particular embodiments of this disclosure include baking soda (e.g., sodium, potassium, or aluminum bicarbonate), baking acid (e.g., sodium aluminum phosphate, monocalcium phosphate, or dicalcium phosphate), and combinations thereof.H. Dairy Products

[0476] In one embodiment, the sweetened composition is a dairy product. Dairy products and processes for making dairy products suitable for use in this disclosure are well known to those of ordinary skill in the art. Dairy products, as used herein, comprise milk or foodstuffs produced from milk. Non-limiting examples of dairy products suitable for use in embodiments of this disclosure include milk, milk cream, sour cream, creme fraiche, buttermilk, cultured buttermilk, milk powder, condensed milk, evaporated milk, butter, cheese, cottage cheese, cream cheese, yogurt, ice cream, frozen custard, frozen yogurt, gelato, via, piima, filmjolk, kajmak, kephir, viili, kumiss, airag, ice milk, casein, ayran, lassi, khoa, or combinations thereof. The dairy products can be produced through conventional means or can be filtered or further modified to adjust the taste properties. In certain embodiments, the dairy products can be liquid dairy products from which one or more of the carbohydrate sugars (lactose or its breakdown products galactose or glucose) are reduced as compared to milk prior to such processing, or are substantially removed and which are supplemented with the sweetening composition described herein. The reduction of carbohydrates can be about 5% or about 10% or about 20% or about 50% or about 70% or more as compared to unprocessed milk.

[0477] According to particular embodiments of this disclosure, the dairy compositions also may comprise other additives. Non-limiting examples of suitable additives include sweeteners as disclosed herein and flavorants such as chocolate, strawberry, and banana. Particular embodiments of the dairy compositions provided herein also may comprise additional nutritional supplements such as vitamins (e.g., vitamin D) and minerals (e.g., calcium) to improve the nutritional composition of the milk.I. Tabletop Sweetener Compositions

[0478] In one embodiment, the sweetened composition is a tabletop sweetener. The tabletop sweetener can further include at least one bulking agent, additive, anti-caking agent, functional ingredient or combination thereof.

[0479] Suitable "bulking agents" include, but are not limited to, maltodextrin (10 DE, 18 DE, or 5 DE), com syrup solids (20 or 36 DE), sucrose, fructose, glucose, invert sugar, sorbitol, xylose, ribulose, mannose, xylitol, mannitol, galactitol, erythritol, maltitol, lactitol, isomalt, maltose, tagatose, lactose, inulin, glycerol, propylene glycol, polyols, polydextrose, fructooligosaccharides, cellulose and cellulose derivatives, and the like, and mixtures thereof. Additionally, in accordance with still other embodiments of the present disclosure, granulated sugar (sucrose) or other caloric sweeteners such as crystalline fructose, other carbohydrates, or sugar alcohol can be used as a bulking agent due to their provision of good content uniformity without the addition of significant calories.

[0480] As used herein, the phrase "anti-caking agent" and "flow agent" refer to any composition which assists in content uniformity and uniform dissolution. In accordance with particular embodiments, non-limiting examples of anti-caking agents include cream of tartar, calcium silicate, silicon dioxide, microcrystalline cellulose (Avicel, FMC BioPolymer, Philadelphia, PA), and tricalcium phosphate. In one embodiment, the anti-caking agents are present in the tabletop sweetener composition in an amount from about 0.001 to about 3% by weight of the tabletop sweetener composition.

[0481] The tabletop sweetener compositions can be packaged in any form known in the art. Non-limiting forms include, but are not limited to, powder form, granular form, packets, tablets, sachets, pellets, cubes, solids, and liquids.

[0482] In one embodiment, the tabletop sweetener composition is a single- serving (portion control) packet comprising a dry-blend. Dry-blend formulations generally may comprise powder or granules. Although the tabletop sweetener composition may be in a packet of any size, an illustrative non-limiting example of conventional portion control tabletop sweetener packets are approximately 2.5 by 1.5 inches and hold approximately 1 gram of a sweetener composition having a sweetness equivalent to 2 teaspoons of granulated sugar (.about.8 g). In a particular embodiment, a dry-blend tabletop sweetener formulation may contain a sweetener an amount from about 1% (w / w) to about 10% (w / w).

[0483] A tabletop sweetener composition also may be embodied in the form of a liquid, wherein a composition of the present disclosure is combined with a liquid carrier. Suitablenon-limiting examples of carrier agents for liquid tabletop sweeteners include water, alcohol, polyol, glycerin base or citric acid base dissolved in water, and mixtures thereof. The sweetness equivalent of a tabletop sweetener composition for any of the forms described herein or known in the art may be varied to obtain a desired sweetness profile. For example, a tabletop sweetener composition may comprise a sweetness comparable to that of an equivalent amount of standard sugar. In another embodiment, the tabletop sweetener composition may comprise a sweetness of up to 100 times that of an equivalent amount of sugar. In another embodiment, the tabletop sweetener composition may comprise a sweetness of up to 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, and 2 times that of an equivalent amount of sugar.J. Delivery Systems

[0484] The presently disclosed sweetener compositions can also be formulated into various delivery systems having improved ease of handling and rate of dissolution. Non-limiting examples of suitable delivery systems comprise sweetener compositions co-crystallized with a sugar or a polyol, agglomerated sweetener compositions, compacted sweetener compositions, dried sweetener compositions, particle sweetener compositions, spheronized sweetener compositions, granular sweetener compositions, and liquid sweetener compositions.IX. Flavor Modulators

[0485] Natural sugars, such as sucrose, fructose and glucose, are utilized in the food and beverage industries to provide a pleasant taste to foods and beverages. In addition, natural sugars are commonly used in pharmaceuticals, nutraceuticals, and oral hygienic / cosmetic products to similarly impart a pleasant taste. Sucrose, in particular, imparts a taste that is highly preferred by many consumers. Although sucrose provides superior sweetness characteristics, it is caloric. High-potency sweeteners have been introduced to address consumer demand for products having a pleasant taste, while at the same time meet increasing demand for healthier, reduced calorie products. Moreover, the demand for healthier, reduced calorie products is being driven by public policy and regulatory mandates.

[0486] However, high potency sweeteners differ significantly from natural caloric sugars in ways that often frustrate consumers and limit market penetration of products containing many high potency sweeteners. On a taste basis, high potency sweeteners exhibit temporal profiles, maximal responses, flavor profiles, mouthfeels, and / or adaptation behaviors that differ from sugar. Commonly, high potency sweeteners exhibit delayed sweetness onset, lingeringsweet aftertaste, bitter taste, metallic taste, astringent taste, cooling taste and / or licorice-like taste. High potency sweeteners may be synthetic chemicals, natural substances, physically or chemically modified natural substances, and / or reaction products obtained from synthetic and / or natural substances. The desire for natural high potency sweeteners with favorable taste characteristics remains high.

[0487] One class of high potency sweeteners are the steviol glycosides. However, utilization has been limited to date by certain undesirable taste properties, including licorice taste, bitterness, astringency, sweet aftertaste, bitter aftertaste, and licorice aftertaste. These undesirable taste properties tend to become more prominent with increased concentration. For example, these undesirable taste attributes are particularly prominent in carbonated beverages, where full replacement of sugar may involve concentrations of steviol glycosides that exceed 500 mg / L.

[0488] Additional components of the presently described sweeteners and sweetened compositions can be included to modulate the flavor of the compositions, as well as the stability, of the compositions, as described in further detail below.

[0489] Bitter blocking agents: bitter blocking flavors work across a range of bitterants, including caffeine. They mitigate bitterness associated with functional ingredients that become more predominant when reducing sugar in health and wellness products. Examples of bitter blocking agents include, but are not limited to, sweeteners such as sugar, honey, fruits, aspartame, monellin, and neotame, salt, herbs, P-cyclodextrin, homoeriodictyol sodium salt, zinc sulfate monohydrate, magnesium sulfate, carboxymethylcellulose sodium salt, gymnemic acids, miraculin, sodium acetate sodium gluconate, adenosine 5 ’-monophosphate, trilobatin and hesperetin dihydrochalcone 4-P-D-glucoside. Linger control agents: linger control agents control the sweet linger during the taste experience, leaving consumers with a desirable balance in sweetness and aftertaste, since high intensity sweeteners used to replace sugar are often associated with a cloying, sweet linger. One example of a linger control agent is chlorhexidine. Textural balance agents: As foods and beverages are consumed, different perceptions are experienced throughout the entire taste profile. How the flavors are released through the tasting experience can rely on the textural balance of the product. Textural balance agents improve texture to craft an always pleasing experience. Examples of textural control agents for stabilization or thickening of compositions include, but are not limited to, hydrocolloids and emulsifiers. Sweetness enhancement agents: Sweetness enhancement agents provide a wide array of natural flavor options for sugar reduced products. Examples of sweetness enhancement agents include, but are not limited to, sucrose, saccharin, aspartame, sucralose,acesulfame potassium, isomalt, lactitol, maltitol, carob, mesquite, lucuma, spices such as vanilla, cinnamon, holy basil, nutmeg and cardamom, aromas such as vanilla, honey and banana, thaumatin, neotame, and positive allosteric modulators. , holy basil, Mouthfeel enhancer agents: When developing a preferred sugar reduced product, mouthfeel is as important as the sweetness. Mouthfeel enhancers provide increased mouthfeel at various levels to deliver enhancement from low sugar reduction up to zero added sugar products. Examples of mouthfeel enhancers include, but are not limited to, starches, such as modified corn starch and tapioca starch, gums and hydrocolloids, such as xanthan gum, guar gum and carrageenan, dairy proteins, such as whey protein concentrate and casein, fat replacers, such as microcrystalline cellulose and inulin, emulsifiers, such as lecithin and mono- and diglycerides, and polydextrose. Astringency reduction agents: In the process of reducing sugar, the sweet to acid ratios can often become unbalanced. Examples of astringency reduction agents include, but are not limited to, milk and milk proteins, egg whites, polyvinylpyrrolidone (PVPP), sucrose, lemon, cooling essential oils such as bergamot, fats, acids, isinglass, and aroma compounds with a sweet connotation. Functional ingredients, such as plant proteins, can exhibit more astringency in the finished product. Astringency reduction agents rebalance food and beverage products to provide the right amount of acidity and sweetness to deliver a low- to-zero calorie option with a great taste.

[0490] The presently disclosed sweetener and sweetened compositions can also comprise stabilization agents, including, but not limited to, carrageenan, guar gum, gellan gum, xanthan gum and mono- and diglycerides.X. Additional Definitions

[0491] The following definitions or interpretations of technical terms will be used throughout the present disclosure. The technical terms used herein are generally to be given the meaning commonly applied to them in the pertinent art of plant biology, molecular biology, bioinformatics, and plant breeding. All of the following term definitions apply to the complete content of this application.

[0492] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one. " The use of the term "or" in the claims is used to mean "and / or" unless explicitlyindicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0493] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." As used herein, the phrase "consisting essentially of" requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0494] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0495] As used herein, words of approximation such as, without limitation, "about," "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to thepreceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0496] The terms "peptides," "oligopeptides," "polypeptide," "protein", or "enzyme" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds, unless mentioned herein otherwise. The terms "gene sequence(s)," "polynucleotide(s)," "nucleic acid sequence(s)," "nucleotide sequence(s)," "nucleic acid(s)," "nucleic acid molecule" are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.

[0497] Endogenous. An "endogenous" or "native" nucleic acid and / or protein refers to a nucleic acid and / or protein as found in a plant or other organism in its natural form (i.e., without there being any human intervention, such as recombinant DNA engineering technology),

[0498] Exogenous. The term "exogenous" (in contrast to "endogenous") means a nucleic acid or protein that has been introduced in a plant or other organism by means of recombinant DNA technology. An "exogenous" nucleic acid or protein can either not occur in a plant in its natural form, be different from the nucleic acid or protein as found in a plant in its natural form, be present at a higher or lower level than the nucleic acid or protein naturally present in a plant, or in the case of a nucleic acid can be identical to a nucleic acid found in a plant in its natural form, but integrated at a location different that its natural genetic environment.

[0499] Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.

[0500] Expression Cassette. A nucleic acid sequence of interest operably linked to one or more control sequences (at least to a promoter) as described herein. An expression cassette can also include additional transcriptional and / or translational enhancers. An expression cassette can also include terminator, silencer and enhancer sequences, intron sequences added to the 5' untranslated region (UTR) or in the coding sequence of the nucleic acid sequence, and / or other control sequences such as protein and / or RNA stabilizing elements. An expression cassette may be integrated into the genome of a host cell and replicated together with the genome of said host cell, or transiently present in a host cell.

[0501] Genetic Transformation: A process of introducing a DNA sequence or construct (e.g., a vector or expression cassette) into a cell or protoplast in which that exogenous DNA is expressed transiently without incorporation into the genome, incorporated into a chromosome or is capable of autonomous replication.

[0502] Heterologous: A sequence that is not normally present in a given host genome in the genetic context in which the sequence is currently found In this respect, the sequence may be native to the host genome, but be rearranged with respect to other genetic sequences within the host sequence. For example, a regulatory sequence may be heterologous in that it is linked to a different coding sequence relative to the native regulatory sequence.

[0503] Modulation. The term modulation refers to when the expression level is changed in comparison to the expression seen in a control plant. Modulation refers to an expression level that is either increased or decreased.

[0504] Obtaining: When used in conjunction with a transiently expressing plant cell or transiently expressing plant tissue, obtaining means either transforming a non-transgenic plant cell or plant tissue to create the transiently expressing plant cell or plant tissue.

[0505] Operably Linked. The term "operably linked" or "functionally linked" is used interchangeably and, as used herein, refers to a functional linkage between, for example, a promoter sequence and a nucleic acid sequence of interest, such that the promoter sequence is able to direct transcription of the nucleic acid sequence of interest, or a functional linkage between a terminator sequence and a nucleic acid sequence of interest, such that the terminator sequence is able to stop or terminate transcription of the nucleic acid sequence of interest.

[0506] Plant. The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including fruits, seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene / nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene / nucleic acid of interest.

[0507] Ploidy. Ploidy or chromosomal ploidy refers the number of complete sets of chromosomes occurring in the nucleus of a cell. Somatic cells, tissues, and individual organisms can be described according to the number of sets of chromosomes present (the "ploidy level"): monoploid (1 set), diploid (2 sets), triploid (3 sets), tetrapioid (4 sets), pentapioid (5 sets), hexapioid (6 sets), heptapioid or septapioid (7 sets), etc. The generic term polyploidy is used herein to describe cells with three or more chromosome sets.

[0508] Promoter: A recognition site on a DNA sequence or group of DNA sequences that provides an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.

[0509] Recombinant. A nucleic acid sequence, expression cassette, genetic construct, or vector comprising a nucleic acid sequence as disclosed herein, or an organism transformed with such nucleic acid sequences, expression cassettes or vectors, created by genetic engineering techniques in which either (a) the sequences of the nucleic acids or a part thereof, or (b) genetic control sequence(s) that is operably linked with the nucleic acid sequence, for example a promoter or terminator, or (c) combinations of (a) and (b), are not located in their natural genetic environment or have been modified and / or inserted artificially by genetic engineering methods.

[0510] Selected DNA: A DNA segment that one desires to introduce or has introduced into a plant genome by genetic transformation.

[0511] Terminator. A DNA control sequence at the end of a transcriptional unit that signals 3’ processing and polyadenylation of a primary transcript and termination of transcription.

[0512] Transformation construct: A chimeric DNA molecule that is designed for introduction into a host genome by genetic transformation. Transformation constructs will often comprise all of the genetic elements necessary to direct the expression of one or more exogenous genes. In particular embodiments of the instant disclosure, it may be desirable to introduce a transformation construct into a host cell in the form of an expression cassette.

[0513] Transformed cell: A cell the DNA complement of which has been altered by the introduction of an exogenous DNA molecule into that cell.

[0514] Transgene: A segment of DNA that is capable of autonomous replication in a host cell and is capable of causing the expression of one or more coding sequences. Exemplary transgenes will provide the host cell, or plant part comprising such host cells, with a novel phenotype relative to the corresponding non-transformed cell or plant part. Transgenes may be directly introduced into a plant by genetic transformation.

[0515] Vector: A DNA molecule designed for transformation into a host cell. Some vectors may be capable of replication in a host cell. A plasmid is an exemplary vector, as are expression cassettes isolated therefrom.EXAMPLES

[0516] The following examples are included to demonstrate illustrative embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute one embodiment of modes for its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 Construction of Expression Cassettes

[0517] Various expression cassettes having different combinations of nucleotide sequences encoding the mogroside pathway enzymes and regulatory elements were constructed. Construction of these expression cassettes was carried out following standard genetic engineering methods. The following expression cassettes were constructed.

[0518] SP3034: This expression cassette was assembled using an e35S promoter operably linked to a Dicot Ruby nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a TBSV pl9 nucleic acid sequence, which is operably linked to a PBI Synthetic terminator.

[0519] SP3684: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0520] SP5425: This expression cassette was assembled using an Enhanced AtEF-lA promoter operably linked to a SgSQEl GC nucleic acid sequence, which is operably linked to a Pea 3 A terminator, a DCMV promoter operably linked to a SgCYP87D18 GC nucleic acid sequence, which is operably linked to a AtUBQ3 terminator, a FSgt PFLt chimeric promoter operably linked to a SgCDS Zm nucleic acid sequence, which is operably linked to a Gmax MYB2 terminator, a Duplic MMV promoter operably linked to a SgCYP72A459vla Zmnucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a SgUGT720-269-l GC nucleic acid sequence, which is operably linked to an AtHSP18.2 terminator, an AtUBQlO promoter operably linked to a SgUGT94-289-l GC nucleic acid sequence, which is operably linked to a CsHSP17.3 terminator, a FuasFSc promoter operably linked to a SgEPH3 GC nucleic acid sequence, which is operably linked to a Potato Ubi3 terminator, a ScBV promoter operably linked to a CltHMGR GC nucleic acid sequence, which is operably linked to an AtTUB9 terminator, a CmYLCV promoter operably linked to a SgMGl GC nucleic acid sequence, which is operably linked to a CsHSP22 terminator, a PCLSV promoter operably linked to a SgUGT720-269-l nucleic acid sequence, which is operably linked to a Pea E9 terminator, a FMV Fit promoter operably linked to a BjHMGS mutant nucleic acid sequence, which is operably linked to an AtNDUFA8 terminator, a FS4 promoter operably linked to a C1GPS1 nucleic acid sequence, which is operably linked to a AtFAD2 terminator, a CsVMV Promoter-SynJ 5' leader promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator.

[0521] SP1463: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0522] SP3139: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to aGmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a e35S promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0523] SP1908: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0524] SP3488: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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, an CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to atHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0525] SP3015: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AIRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0526] SP3432: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to a 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, a 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, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, an e35S promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0527] SP1160: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronicnucleic acid sequence, which is operably linked to a AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0528] SP2916: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0529] SP4643: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQEnucleic acid sequence, which is operably linked to a Pea3A terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0530] SP4870: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0531] SP1603: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked toan ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0532] SP3095: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0533] SP0265: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to a SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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 UGT720:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0534] SP4406: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence,which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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 a AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0535] SP2152: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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 a AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0536] SP4311 : This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoteroperably 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, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScB V promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0537] SP4378: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0538] SP3132: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, aNOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0539] SP2355: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0540] SP4762: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by an e35S promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to a AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoteroperably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0541] SP0892: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to a ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0542] SP2249: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0543] SP0796: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLtpromoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScB V promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0544] SP2057: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0545] SP3308: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence,which is operably linked to an Ubi3 terminator, a ScB V promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0546] SP1379: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0547] SP3494: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0548] SP2585: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to a SQE nucleic acid sequence,which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, an e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0549] SP3635: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to a 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, a 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 UGT720:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0550] SP3800: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to a SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, aNOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, an e35S promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0551] SP0981: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0552] SP0137: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0553] SP2154: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0554] SP1727: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0555] SP0075: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence,which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0556] SP4305: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0557] SP4221 : This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked toan SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0558] SP3488: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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, an CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a dMMV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0559] SP4094: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to anATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0560] SP2971: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0561] SP2049: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operablylinked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0562] SP4063: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0563] SP0121 : This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0564] SP3358: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to an SQE nucleic acid sequence,which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, an e35S promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0565] SP4513: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0566] SP2221 : This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72:2A:CYP72bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0567] SP3925: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0568] SP3748: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked toan ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0569] SP4511: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0570] SP3547: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0571] SP3481 : This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0572] SP2185: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0573] SP2792: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV promoter operably linked to an SQE nucleic acid sequence,which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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 UGT720:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0574] SP1000: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0575] SP3766: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a 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, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to aUGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0576] SP4353: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0577] SP0255: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoteroperably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0578] SP4815: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH 12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0579] SP1073: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronic nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a 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 e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0580] SP4402: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to a CYP72:2A:CYP72 bicistronicnucleic acid sequence, which is operably linked to an AtRBCS2B terminator (all in reverse orientation), a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScB V promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0581] SP1415: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0582] SP2353: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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 bicistronicnucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0583] SP0565: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FMVSgt 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, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScB V promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0584] SP1202: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FMVSgt 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, an e35S promoter operably linked to a UGT720:2A:UGT720 bicistronic nucleic acid sequence, which is operably linked to an E9 terminator, a CmYLCV promoter operably linked to a UGT94:2A:UGT94 bicistronic nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoteroperably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE3 promoter operably linked to a tHMGR:2A:tHMGR bicistronic nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0585] SP2808: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub89 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0586] SP4522: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a 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 Zm nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0587] SP3842: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35 S terminator, followed by a TM6 MAR insulator sequence.

[0588] SP3938: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0589] SP3318: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLtpromoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CmYLCV promoter operably linked to a UGT74_3 nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0590] SP3493: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CmYLCV promoter operably linked to a UGT74_4 nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0591] SP2476: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D20 m2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, aFSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0592] SP4983: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D20 m3 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0593] SP4003: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D17 m2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acidsequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0594] SP4074: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D17 m3 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0595] SP2649: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D18_B m3 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35Sterminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an A1UBQ3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0596] SP3397: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D17 m2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a LJGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CmYLCV promoter operably linked to a UGT74_3 nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0597] SP2771: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D17 m3 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which isoperably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CmYLCV promoter operably linked to a UGT74_3 nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0598] SP0847: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 CYP87D17 m3 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a LJGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a DCMV promoter operably linked to a SgCPR2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a CmYLCV promoter operably linked to a UGT74_4 nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0599] SP3468: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a FE_3 promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a FE_3 promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a 35S terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a CmYLCV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a FS1_1 promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to anUbi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0600] SP2177: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a FS 1_1 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a 35S terminator, a FE_3 promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to a 35S terminator, a FE_3 promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a 35S terminator, a FE_3 promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an 35S terminator, a FE_3 promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, a FE_3 promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an 35S terminator, a FS1_1 promoter operably linked to an EPH nucleic acid sequence, which is operably linked to a 35S terminator, a FS1_1 promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to a 35S terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0601] SP3804: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3A terminator, an e35S promoter operably linked to a CYP87 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a ATHSP18.2 terminator, an e35S promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a e35S promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0602] SP3016: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a HLVH12 promoter operably linked to an SQE nucleic acid sequence, which is operably linked to a Pea3 A terminator, a DCMV promoter operably linked to a C YP87 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to aGmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0603] SP0036: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 e35S promoter operably linked to a CDS nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0604] SP1458: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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 e35S promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to a 35S terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0605] SP0336: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a dMMV promoter operably linked to an green fluorescent protein nucleic acid sequence, which is operably linked to a PBI terminator, a PCLSV promoter operably linked to an SQE-2 nucleic acid sequence, which is operably linked to a Pea3A terminator, a DCMV promoter operably linked to a CYP87-2 nucleic acid sequence, which is operably linked to an AtUBQ3 terminator, a Fsgt / PFLT promoter operably linked to a CDS-2 nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a CsVMV promoter operably linked to an EPH-2 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a HLVH12 promoter operably linked to a UGT720-2 nucleic acid sequence, which is operably linked to an E9 terminator, a FMVSgt promoter operably linked to an EPH-2 nucleic acid sequence, which is operably linked to an Ubi3 terminator, an e35S promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0606] SP0315: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV 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, a Fgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a HLVH12 promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a CsVMV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a 35S promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0607] SP3190: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0608] SP3029: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a ScBV promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0609] SP0545: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a CmYLCV promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, an e35S promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a FE_3 promoter operably linked to a tHMGR nucleic acid sequence, which is operably linked to an AtTub terminator, a CsVMV promoter operably linked to a HygR nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0610] SP4156: This expression cassette was assembled using a TM6 MAR insulator sequence, followed by a CmYLCV 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, a Fgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a HLVH12 promoter operably linked to a UGT720 nucleic acid sequence, which is operably linked to an E9 terminator, a CsVMV promoter operably linked to a UGT94 nucleic acid sequence, which is operably linked to an ATHSP18.2 terminator, a NOS promoter operably linked to an EPH nucleic acid sequence, which is operably linked to an Ubi3 terminator, a 35S promoter operably linked to a nptll nucleic acid sequence, which is operably linked to a 35S terminator, followed by a TM6 MAR insulator sequence.

[0611] SP5570: This expression cassette was assembled using an HLVH12 promoter operably linked to an SgSQEl 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, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, an e35S promoter operably linked to a SgUGT720-269-l GC nucleic acid sequence, which is operably linked to an AtHSP18.2 terminator, an AtUBQlO promoter operably linked to a SgUGT94-289-l GC nucleic acid sequence, which is operably linked to a CsHSP17.3 terminator, a FuasFSc promoter operably linked to a SgEPH3 GC nucleic acid sequence, which is operably linked to a Potato Ubi3 terminator, a ScBV promoter operably linked to a CltHMGR GC nucleic acid sequence, which is operably linked to an AtTUB9 terminator, a CmYLCV promoter operably linked to a SgMGl GC nucleic acid sequence, which is operably linked to a CsHSP22 terminator, a PCLSV promoter operably linked to a S1UGT4 GC nucleic acid sequence, which is operably linked to a Pea E9 terminator, a dMMV promoter operably linked to a DsRed nucleic acid sequence, which is operably linked to an AtuG7 terminator, a CsVMV Promoter-SynJ 5' leader promoter operably linked to a NPTII nucleic acid sequence, which is operably linked to a 35 S terminator..

[0612] SP5834: This expression cassette was assembled using an HLVH12 promoter operably linked to an SgSQEl nucleic acid sequence, which is operably linked to a Pea3A terminator, a DCMV promoter operably linked to a CYP87 nucleic acid sequence, which isoperably linked to an AtUBQ3 terminator, a FSgt / PFLt promoter operably linked to a CDS nucleic acid sequence, which is operably linked to a GmaxMYB2 terminator, a dMMV promoter operably linked to a CYP72 Zm nucleic acid sequence, which is operably linked to an AtRBCS2B terminator, a potato Ubi promoter operably linked to a SgUGT720-269-l GC nucleic acid sequence, which is operably linked to an AtHSP18.2 terminator, an AtUBQlO promoter operably linked to a SgUGT94-289-l GC nucleic acid sequence, which is operably linked to a CsHSP17.3 terminator, a potato UBQ promoter operably linked to a SgEPH3 GC nucleic acid sequence, which is operably linked to a Potato Ubi3 terminator, a BvMIl promoter operably linked to a CltHMGR GC nucleic acid sequence, which is operably linked to an AtTUB9 terminator, a CmYLCV promoter operably linked to a SgMGl GC nucleic acid sequence, which is operably linked to a CsHSP22 terminator, a BvUBQ promoter operably linked to a PgtHMGR nucleic acid sequence, which is operably linked to a Pea E9 terminator, a BvMIl promoter operably linked to a SgUGT720-269-l / SgUGT94-289-l fusion protein GC nucleic acid sequence, which is operably linked to an AtFAD2 terminator, a dMMV promoter operably linked to a DsRed nucleic acid sequence, which is operably linked to an AtuG7 terminator, a CsVMV Promoter-SynJ 5' leader promoter operably linked to a NPTH nucleic acid sequence, which is operably linked to a 35S terminator.

[0613] BvCYP76ADl: This expression vector was assembled using a CmYLCV promoter operably linked to a BvCYP76ADl nucleic acid sequence, which is operably linked to a cucumber HSP17.3 terminator.

[0614] BvCYP76AD6: This expression vector was assembled using a CmYLCV promoter operably linked to a BvCYP76AD6 nucleic acid sequence, which is operably linked to a cucumber HSP22 terminator.

[0615] BvDODAl: This expression vector was assembled using a CaMV e35S promoter operably linked to a BvDODAl nucleic acid sequence, which is operably linked to an Arabidopsis HSP18.2 terminator.

[0616] MjcDOPA5GT: This expression vector was assembled using an Arabidopsis UBQ10 promoter operably linked to a MjcDOPA5GT nucleic acid sequence, which is operably linked to a cucumber HSP22 terminator.

[0617] SP5500: This expression vector was assembled using a CaMV e35S promoter operably linked to a barley alpha-amylase signal peptide, which is operably linked to a human lactoferrin nucleic acid sequence, which is operably linked to a plant ER retention signal, which is operably linked to an Arabidopsis HSP18.2 terminator.

[0618] SP6195: This expression vector was assembled using a CaMV e35S promoter operably linked to a barley alpha-amylase signal peptide, which is operably linked to a bovine lactoferrin nucleic acid sequence, which is operably linked to a plant ER retention signal, which is operably linked to an Arabidopsis HSP18.2 terminator.

[0619] SP5221: This expression vector was assembled using a CaMV e35S promoter operably linked to an eGFP nucleic acid sequence, which is operably linked to a plant ER retention signal, which is operably linked to an Arabidopsis HSP18.2 terminator.Example 2 Preparation of Agrobacterium Solutions for Infiltration

[0620] Agrobacterium infiltration, or “Agroinfiltration,” of plant leaves has been exploited for the commercial production of various proteins, antibodies, and other molecules of interest (Chincinska et al., Plant Meth. 17:83, 2021 ; Tyurin et al., Plants (Basel) 9:1187, 2020; Chen et al., Adv. Tech. Biol. Med. 1: 103, 2013). The most frequently used and robust systems for this purpose have been the leaves of Nicotiana benthamiana or lettuce plants (Chen et al., Meth. Mol. Biol. 1385:55-67, 2016). Typically, leaves of young plants are infiltrated or infused with a solution of Agrobacterium cells containing an expression vector that directs the high- level synthesis of one or more proteins of interest. The efficiency of bacterial penetration and infection of plants can be enhanced for large-scale production by applying vacuum pressure to leaves submerged in the Agrobacterium solution. A lower throughput method often used in research involves the infiltration of bacterial solutions into the underside of leaves using a needle-less syringe.

[0621] The use of this rapid Agrobacterium infiltration method using tissues other than leaves has not been reported as a viable option for commercial production of molecules of interest. Production of reporter enzymes, such as beta-glucuronidase, has been demonstrated in some non-leaf tissues such as apple, tomato, and strawberry fruits (Lv et al., 2019; Hoshikawa et al., 2019; Zhao et al., 2019), but the true efficiency of these production systems is difficult to assess due to the diffuse blue staining produced by the highly stable GUS enzyme that occurs only over a long period of time. Thus, one cannot predict , based on currently available information, whether these systems described by Lv et al., Scientia Horticulturae Vol. 256, 2019; Hoshikawa et al., Plant Cell. Rep. 38:75-84, 2019; Zhao et al., Hortic. Res. 6:53, 2019 could be used for large-scale manufacturing of desired molecules with high reproducibility and favorable economics.

[0622] The inventors surveyed a wide variety of fruit and vegetable tissues for their ability to be efficiently infected by Agrobacterium, using either small tissue cores or as intact fruits. Unexpectedly, the majority of tissues that were tested did not produce any visible pigment when testing enzymes expressed from the RUBY reporter vector described by He et al. (Hortic. Res. 7 : 152, 2020). Additional tests were performed for certain tissues using thin slices soaked in Agrobacterium cell suspensions, but these also failed to show any clear evidence of bacterial infection or reporter enzyme production. The rare exceptions for the tissue core method were immature watermelon fruits, which often had very high reporter gene activity, and potato tuber and sugar beet taproot slices, which had random, small areas of reporter gene expression.

[0623] Surprisingly, the inventors found that intact fruits harvested from certain plants were much more amenable to the new infiltration method described below in Examples 4 and 5 and were able to synthesize significant amounts of desirable end-products. Intact fruits were infiltrated with Agrobacterium strains designed to express the genes coding for betalain pigments (RUBY) and non-caloric mogroside sweetener molecules (Itkin et al., Proc. Natl. Acad. Sci. USA 113:E7619-E7628, 2016). Intact zucchini and immature watermelon fruits produced high levels of betalain pigments and non-caloric mogroside sweetener molecules following infiltration with the relevant Agrobacterium strains. Most of the other intact fruits that were evaluated did not show any significant evidence of production of the betalain pigments or non-caloric mogroside sweetener molecules. The ability to use these two readily available fruits with the Agrobacterium infiltration method described in Examples 4 and 5 opens up new commercial opportunities for the rapid production of high-value compounds in plant cells.

[0624] Binary plant transformation vectors for production of betalain pigments (vector SP3034 - RUBY; see Example 1) or mogroside sweeteners (vectors SP3684, SP5425, SP5570 or SP5834; see Example 1) were electroporated into competent EHA105 Agrobacterium cells. Colonies containing the correct vectors were identified by extraction of plasmid DNA from Agrobacterium liquid cultures and evaluation by restriction enzyme digestion. Vector maps for SP3034, SP3684, SP5425, SP5570 and SP5834 are shown in FIG. 2, FIG. 3, FIG. 4, FIG. 5 and FIG. 6, respectively.

[0625] Agrobacterium cells containing the vectors of interest were grown overnight with shaking at 28°C in 3 ml of YEP media + 50 pg / ml kanamycin (or 100 pg / ml spectinomycin for SP5570) + 25 pg / ml rifampicin. The next day, the 3 ml culture was transferred into a 250 ml flask containing 40 ml of YEP media + 50 pg / ml kanamycin (or 100 pg / ml spectinomycin for SP5570) + 25 pg / ml rifampicin and grown with shaking overnight at 28°C. On day 3, theAgrobacterium cells were pelleted by centrifugation at 4150 rpm for 15 minutes at room temperature. The supernatant was decanted and then the bacterial pellet was resuspended by vortexing in 20 ml of Infiltration Buffer 1 (10 mM MES, 10 mM MgC12, pH 5.7) or Infiltration Buffer 2 (10 mM sodium citrate, 10 mM MgCh, pH 5.7). The optical density of a 1 : 10 dilution of the resuspended cells was measured using a spectrophotometer, and the final density of the Agrobacterium cell suspension to be infiltrated was diluted to the desired level (typically OD600 = 1.0 to 1.6). Acetosyringone was added to the Agrobacterium solution at a final concentration of 200 M, and the bacterial cells were incubated for at least 2 hours to overnight for induction of the virulence genes.Example 3 Analysis of Mogroside Compounds

[0626] Samples were prepared for LC-MS / MS analysis of mogrosides by 1) flash freezing in liquid nitrogen, 2) freeze-drying in an FRZ-5193 (Price’s Scientific Services Inc.), and 3) grinding the freeze-dried material into a powder with a SPEX SamplePrep 1600 MiniG bead beater. Powders were then extracted with an 80% (w / w) methanol / water solution for 5 seconds with vortexing, followed by centrifugation at 4198 RCF for 20 minutes at 4°C. The supernatant was then filtered through VWR 1.5 ml 0.2 pm low protein binding modified nylon fdters and centrifuged for 5 minutes at 1383 RCF.

[0627] The initial liquid chromatographic (LC) separation of the samples was done using a Waters Quaternary Solvent Manager ACQUITY UPLC H-Class PLUS connected to a Waters Sample Manager FTN-H ACQUITY UPLC and a PDA eX Detector ACQUITY UPLC. The LC gradients use an ACQUITY UPLC CSH Cl 8 1.7 pm 2.1 mm x 100 mm column at 35°C with an acetonitrile / water gradient acidified with 0.1% formic acid. The LC system was coupled to an Xevo G2-XS mass spectrometer in sensitivity positive mode using the MSMS scan mode with a collision energy of 30 for mogrosides and 11 -oxo-mogrosides with five, three, or two glucose and collision energy of 20 for mogrosides and 11 -oxo-mogrosides with one glucose or mogrol. The following precursor and product ion pairs, expressed in m / z, were used to quantify the mogrosides with a 50 mDa window applied to the product ion: mogrosides with five glucose (1287.7 / 423.3621), mogrosides with four glucose (1125.6 / 423.3621), mogrosides with three glucose (963.6 / 423.3621), mogrosides with two glucose (801.5 / 423.3621), mogrosides with one glucose (639.4 / 423.3621), mogrol (477.4 / 423.3621), 11 -Oxo-mogrosides with five glucose (1285.7 / 457.3680), 11 -Oxo-mogrosides with four glucose (1123.6 / 457.3680), 11 -Oxo-mogrosides with three glucose (961.5 / 457.3680), and 11-Oxo-mogrosides with two glucose (799.5 / 457.3680), and 11-Oxo-mogrol (475.4 / 457.3680). For LockSpray mass correction, 200 pg / ml leucine-enkephalin monitoring for 556.2771 m / z was used. Data analysis was done using the Waters TargetLynx software, excel, R, and JMP.Example 4 Agrobacterium Infection of Fruit and Vegetable Tissues

[0628] Various fruits and vegetables were purchased from a local supermarket and evaluated for their ability to be infected by Agrobacterium cells containing the RUBY reporter gene. Tissue cores were prepared from each plant source using a #5 cork borer and placed into a 24- well microtiter plate (4 replicates per source). A cell suspension of the Agrobacterium strain induced with 200 pM acetosyringone was injected into each of the tissue cores multiple times using a 1 ml syringe with a 5 / 8” x 25-guage needle. The microtiter plate was incubated in the dark at 26°C for 5-7 days and the cores were observed for any red pigmentation due to expression the reporter gene. The only tissue cores that show strong red pigmentation were derived from immature watermelon fruits (11-13 days after flower fertilization). Some small areas of red color were also observed on the cores derived from potato tubers. The results are shown in Table 1.Table 1Example 5Alternative Method for Agrobacterium Infection of Fruit and Vegetable Tissues

[0629] An alternative strategy was evaluated in which various fruits purchased from a local supermarket were tested for their ability to express the RUBY reporter gene for betalain pigments and the genes for the non-caloric mogroside sweetener molecules, following infiltration with the relevant Agrobacterium strains. Agrobacterium cell suspensions induced with 200 pM acetosyringone was injected directly into each of the intact fruits multiple times using a 3 ml syringe with a 1.5” x 18-guage needle. The injected fruits were placed inside a plastic box with the lid kept sightly open to maintain modest humidity, and also covered with foil to provide partial darkness. In addition, certain fruits or tubers were sliced into thin sections and soaked in Agrobacterium solution for 30 minutes, and then placed in petri dishes containing 1 / 2X MS salts in 1.6% solidified agar. The fruits or thin slices were left at 26°C for 5-7 days. The thin slices were visually inspected for evidence of red color resulting from RUBY gene expression, and the red tissue was excised and tested for the presence of mogrosides using the methods described in Example 3. The intact fruits were cut open with a knife and visually inspected for evidence of red color resulting from RUBY gene expression, after which the red tissue was excised and tested for the presence of mogrosides using the methods described in Example 3.

[0630] Intact zucchini, cucumber, and immature watermelon fruits produced strong areas of red pigmentation and high levels of mogroside sweetener molecules (Table 2). Intact tomato fruits, acorn squash, and prickly pear cactus fruits produced areas of red pigment and lower levels of mogrosides. Thin slices of potato tubers and sugar beet taproots also had some areas of red and very low levels of mogrosides in these isolated spots. In contrast, many of the other fruits tested did not produce any red pigment. Thin tissue slices from zucchini and cucumber also did not appear to make red pigment at any significant level, even though these fruits worked the best when Agrobacterium solution was directly injected into intact fruit. These results illustrate the potential of transient expression in fruits as a production system for molecules of interest, and show the novel and unexpected result that high production performance only results when applying the novel infiltration methods reported herein.Table 2Example 6Production of Mogroside Compounds in Transiently Infiltrated Intact Immature Watermelon Fruits

[0631] Whole immature watermelon fruits (9-12 days old) were syringe-infiltrated with an Agrobacterium solution prepared as in Example 2 with Infiltration Medium 2 that contained a mixture of SP5834 (OD600 = 1.0) + SP5425 (OD600 = 0.5) + SP3034 (OD600 = 0.3). Approximately 1 / 10 volume of Agrobacterium solution (in ml) was injected per gram of fruit fresh weight. The infiltrated fruits were incubated at room temperature in a covered container for 12 days, and then cut open to harvest mesocarp samples. Small pieces with the strongest RUBY color were collected as ‘surgical samples,’ and the entire mesocarp was isolated, weighed, and blended with 1 volume of hot water using a NutriBullet® blender (2X for 30 sec). The slurry was heated at 90 DC for 20 minutes, and then the solids were pelleted by centrifugation for 12 minutes at 4125 rpm. A 200 pl aliquot of the clarified juice (supernatant)was transferred to a new tube for direct extraction with 800 l of 100% MeOH and subsequent mogroside analysis as outlined in Example 3.

[0632] As indicated in FIG. 7, the surgical samples from 6 independent fruits ranged from approximately 10-15 mg / g DW of total mogrosides, with the majority of this being Mogroside V. In addition to MV, also detected were a smaller amount of 11 -oxo- mogroside V, various ‘other sweet mogrosides’ (such as MIV, MIV-A, Mill, MIII-E, MIII-A2, MIII-A1), and an even smaller percentage of ‘non-sweet mogrosides’ (such as MII-A2, 0-MII-A2 and MII-A1). As indicated in FIG. 8, this equated to roughly 200-300 PPM of total mogrosides in the juice samples prepared from mesocarp tissue. These results indicate that intact immature watermelon fruits are efficient at producing Mogroside V and 11-oxo-Mogroside V as the primary end-products with the vectors that were introduced.Example 7Production of Mogroside Compounds in Transiently Infiltrated Intact Zucchini Fruits

[0633] Whole zucchini fruits (variety ‘Respect’) from greenhouse-grown plants were placed at 4°C for 2 days and then used for Agrobacterium infiltration. Agrobacterium solution was prepared as in Example 2 with Infiltration Medium 2 containing a mixture of SP5834 (OD600 = 1.0) + SP5425 (OD600 = 0.5) + SP3034 (OD600 = 0.3). Approximately 1 / 10 volume of Agrobacterium solution (in ml) was injected per gram of fruit fresh weight. The infiltrated fruits were incubated at room temperature in a covered container for 11 days, and then cut open to harvest mesocarp samples.

[0634] As shown in FIG. 9, the concentration of total mogrosides in 8 independent fruits ranged from 13-42 mg / g DW, with the majority of the mogrosides being Mogroside V. In addition to MV, also detected were a smaller amount of 11 -oxo-mogroside V, an even smaller amount of various ‘other sweet mogrosides’ (such as MIV, MIV-A, Mill, MIII-E, MIII-A2, MIII-A1), and a tiny percentage of ‘non-sweet mogrosides’ (such as MII-A2, 0-MII-A2 and MII-A1). These results indicate that intact zucchini fruits are very efficient at producing Mogroside V as the primary end-product with the vectors that were introduced.Example 8Production of Betalain Compounds in Transiently Infiltrated Whole Zucchini Fruits

[0635] Betalains are a class of water-soluble pigments found in plants of the order Caryophyllales, where they replace anthocyanin pigments. They are divided into two main categories: betacyanins, which produce red-violet colors, and betaxanthins, which produceyellow-orange colors. Betalains are derived from the amino acid tyrosine and are structurally and chemically distinct from anthocyanins. The most well-known source of betalains is the red beetroot Beta vulgaris), but they are also found in other plants such as amaranth, prickly pear cactus, and pitaya...

Claims

CLAIMS1. A method for producing a compound in a fruit, vegetable or leaf tissue, or a portion thereof, comprising: a) transforming the fruit, vegetable or leaf tissue, or a portion thereof, by contacting the fruit, vegetable or leaf tissue, or portion thereof, with a suspension of Agrobacterium cells comprising an expression construct comprising at least a first nucleic acid sequence encoding at least a first amino acid sequence capable of synthesizing the compound operably linked to a promoter; b) incubating the transformed fruit, vegetable or leaf tissue, or a portion thereof, for between about 3 and about 14 days; and c) isolating the compound.

2. The method of claim 1 , wherein the compound is at least a first mogroside, betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP).

3. The method of claim 2, wherein the compound is at least a first mogroside.

4. The method of claim 3 wherein the at least a first mogroside is mogroside II A, mogroside II Al, mogroside II A2, mogroside II E, 1 1-oxo-mogroside II, mogroside III, mogroside III Al, mogroside III A2, mogroside III E 11-oxo-mogroside III, mogroside IV, mogroside IV A, 11 -oxo-mogroside IV, siamenoside I, mogroside V, 11 -oxo-mogroside V or mogroside VI, or an isomer thereof.

5. The method of claim 1, wherein the fruit, vegetable or leaf tissue, or a portion thereof, is transformed by injecting, dipping, spraying or pressure delivering to the fruit, vegetable or leaf tissue, or a portion thereof, with the Agrobacterium cells.

6. The method of claim 1, wherein the Agrobacterium cells are induced with acetosyringone.

7. The method of claim 1, wherein the at least a first nucleic acid sequence is transiently expressed.

8. The method of claim 1, wherein the fruit, vegetable or leaf tissue is an intact fruit, vegetable or leaf.

9. The method of claim 1 , wherein the fruit, vegetable or leaf tissue is a fruit, vegetable or leaf that has been sliced.

10. The method of claim 1, wherein the fruit, vegetable or leaf tissue is zucchini fruit, cucumber fruit, watermelon fruit, immature watermelon fruit, acorn squash, prickly pear cactus fruit, potato tuber or sugar beet.

11. The method of claim 1 , wherein the fruit, vegetable or leaf tissue, or a portion thereof, is incubated for between about 5 and about 7 days.

12. The method of claim 1, wherein the promoter is a heterologous promoter.

13. The method of claim 12, wherein the heterologous promoter is an inducible, plant, bacterial, viral, synthetic, constitutive, tissue specific, developmentally regulated, cell cycle regulated, temporally regulated, spatially regulated, and / or spatio-temporally regulated promoter.

14. The method of claim 1, wherein the at least a first nucleic acid sequence is operably linked to a terminator.

15. The method of claim 14, wherein the terminator is a heterologous terminator.

16. The method of claim 1, wherein the nucleic acid sequence further comprises a polynucleotide sequence encoding a screenable marker sequence.

17. The method of claim 16, wherein the screenable marker is betalain.

18. A composition comprising a compound produced by the method of claim 1.

19. The composition of claim 18, wherein the compound is at least a first mogroside, betalain, lactoferrin, or a fluorescent protein, such as green fluorescent protein (GFP).

20. The composition of claim 19, wherein the compound is at least a first mogroside.

21. The composition of claim 20 wherein the at least a first mogroside is mogroside II A, mogroside II Al, mogroside II A2, mogroside II E, 11-oxo-mogroside II, mogroside III, mogroside III Al, mogroside III A2, mogroside III E 11-oxo-mogroside III, mogroside IV, mogroside IV A, 11 -oxo-mogroside IV, siamenoside I, mogroside V, 11 -oxo-mogroside V or mogroside VI, or an isomer thereof.

22. A processed lower calorie food or beverage product produced from the composition of claim 18.

23. A method for transient expression of a nucleic acid sequence of interest in a fruit, vegetable or leaf tissue, or a portion thereof, comprising transforming the fruit, vegetable, leaf, or portion thereof, by contacting the fruit, vegetable or leaf tissue, or a portion thereof, with asuspension of Agrobacterium cells comprising an expression construct comprising the nucleic acid sequence of interest, and incubating the transformed fruit, vegetable or leaf tissue, or a portion thereof, for between about 3 and about 14 days.

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