stevia plant, seed, tissue, tissue culture or cultured plant cell, methods for producing a stevia pl

专利摘要:
The present invention provides a non-transgenic stevia plant body rich in M rebaudioside type, which contains M rebaudioside in a large amount compared to a wild type stevia species. The present invention also provides: a method for producing the non-transgenic stevia plant body rich in rebaudioside M; and dry leaves produced from a plant body.
公开号:BR112020006485A2
申请号:R112020006485-8
申请日:2018-10-12
公开日:2020-10-13
发明作者:Kazunari Iwaki；Katsuro Miyagawa；Tadayoshi HIRAI；Naoko Okitsu；Saori TAKEYAMA
申请人:Suntory Holdings Limited；
IPC主号:

专利说明:

[001] [001] The present invention relates to a stevia plant with a high content of rebaudioside M. TECHNICAL FUNDAMENTALS
[002] [002] In response to the diverse needs of consumers, several drinks have been developed and are commercially available. Saccharides, like sucrose, are components very commonly mixed in drinks for the purpose, for example, of imparting sweetness. However, its influence on health due to excessive consumption has been pointed out. Thus, there are increasing needs for less calorie and naturally derived sweeteners. For example, Literature associated with Patent 1 describes a functional sweetener composition containing a vitamin, a high intensity sweetener, and a composition for improving sweetness.
[003] [003] Rebaudioside (hereinafter, also referred to as "Reb") is known as a sweet component contained in a stevia extract. Stevia extract is obtained by extracting and purifying it from a stevia leaf. Stevia is a perennial plant in the Asteraceae family with
[004] [004] In these circumstances, a stevia plant containing 0.38% by weight of rebaudioside M and 3.28% by weight of rebaudioside D per dry leaf is known (Literature associated with patent 3).
[005] [005] [Literature associated with patent 1] JP 2009-517043 À [Literature associated with patent 2] National publication of international patent application 2016-515814 [Literature associated with patent 3] US2016 / 0057955A1
[006] [006] Rebaudioside M supposedly has good flavor quality, among steviol glycosides, but cannot be obtained in large quantities from natural stevia plants. Thus, obtaining it is a cause for concern. Means for solving problems
[007] [007] The present invention provides a genetically unmodified stevia plant with a high M rebaudioside content, containing high M rebaudioside compared to the wild type stevia species, a method of producing the plant, and a method of selecting the plant .
[008] [008] Specifically, the present invention provides the following.
[009] [009] [1-1] A non-genetically modified stevia plant with a high M rebaudioside content comprising 2% or more M rebaudioside in relation to the amount of total steviol glycoside contained in a leaf.
[0010] [0010] [1-2] The plant according to [1-1], additionally comprising 9.5% or more of rebaudioside D with respect to the amount of total steviol glycoside contained in a leaf.
[0011] [0011] [1-3] The plant according to [1-1] or [1-2] with at least one of the following genomic characteristics (1) to (5).
[0012] [0012] (1) Homozygous for the allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T.
[0013] [0013] (2) Homozygous for the allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T.
[0014] [0014] (3) Homozygous for the allele in which the base in the position corresponding to position 41 of SEQ ID NO: 39 is C.
[0015] [0015] (4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
[0016] [0016] (5) Homozygous for the allele in which the base in the position corresponding to position 50 of SEQID NO: 43 is A.
[0017] [0017] [1-4] The plant according to any one of [1-1] to [1-3] where the plant is positive for at least one polymorphic marker selected from the group consisting of PO1 to POS5 .
[0018] [0018] [1-5] A seed, tissue, tissue culture or plant cell grown from the plant according to any of [1-1] to [1-
[0019] [0019] [1-6] The tissue, tissue culture or plant cell grown according to [1-5], which is an embryo, a meristematic cell, a pollen, a leaf, a root, an apex of root, petal, protoplasty, leaf section or callus.
[0020] [0020] [1-7] A method for producing a stevia plant with a high M rebaudioside content, comprising 2% or more M rebaudioside with respect to the amount of total steviol glycoside contained in a leaf, the method comprising a step of crossing a stevia plant, according to any one of [1-1] to [1-4], with a second stevia plant.
[0021] [0021] [1-8] The method according to [1-7], wherein the second plant is the stevia plant according to any one of [1-1] to [1-4].
[0022] [0022] [1-9] An extract of the plant according to any one of [1- 1] to [1-4], the seed, tissue, tissue culture or cell according to [1-5].
[0023] [0023] [1-10] A food or drink product, a sweetening composition, a flavor or a medicine comprising the extract according to [1-9].
[0024] [0024] [1-11] A method for producing an extract containing rebaudioside M, comprising a step of obtaining an extract from the plant according to any one of [1-1] to [1-4], from the seed, tissue, tissue culture or cell according to [1-5].
[0025] [0025] [1-12] A method for producing M rebaudioside, comprising a step of purifying M rebaudioside from an extract containing M rebaudioside according to [1-11].
[0026] [0026] [1-13] A method for producing a food or drink product, a sweetening composition, a flavor or a medicine, comprising a step of mixing an extract obtained by the method according to [1-11], and / or rebaudioside M obtained by the method according to [1-12] with other components.
[0027] [0027] [1-14] A method for selecting a stevia plant with a high M rebaudioside content, comprising a step of detecting the presence and / or absence of at least one of the following genomic characteristics (1) to (5) from the genome of a test plant.
[0028] [0028] (1) Homozygous for the allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T.
[0029] [0029] (2) Homozygous for the allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T.
[0030] [0030] (3) Homozygous for the allele in which the base in the position corresponding to position 41 of SEQID NO: 39 is C.
[0031] [0031] (4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
[0032] [0032] (5) Homozygous for the allele in which the base in the position corresponding to position 50 of SEQID NO: 43 is A.
[0033] [0033] [1-15] The method according to [1-14], comprising a step of detecting at least one polymorphic marker selected from the group consisting of PO1 to POS from the genome of a test plant.
[0034] [0034] [1-16] The method according to [1-14] or [1-15], further comprising a step of measuring the content of rebaudioside M in a leaf tissue.
[0035] [0035] [1-17] Any one or more primer oligonucleotide set (s) selected from the group consisting of: (1) a primer oligonucleotide set comprising a primer oligonucleotide sense with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 2; (2) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 3 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 4; (3) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 5 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 6; (4) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 7 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 8; and (5) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 9 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 10, in which the sequence of any 15 or more consecutive bases is positioned at the 3rd end of each primer oligonucleotide.
[0036] [0036] [1-I8] A kit comprising a primer oligonucleotide set according to [1-17] and optionally a restriction enzyme, wherein in the case of the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 contiguous bases or more in SEQ ID NO: 1, the restriction enzyme comprises Xbal; in the case the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 3, the restriction enzyme comprises Kpnl; in the case that the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 5, the restriction enzyme comprises AfIII; and in the case that the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 9, the restriction enzyme comprises Pvul.
[0037] [0037] [1-1] A probe comprising a nucleotide sequence shown in any one of SEQ ID NOs: 55 to 64, the probe being optionally linked with a detectable tag.
[0038] [0038] [1-20] The probe according to [1-19], in which the probe has a fluorescent label, a dye or a binding fraction.
[0039] [0039] [1-21] A method for selecting a genetically unmodified stevia plant with a high M rebaudioside content, comprising steps of: performing PCR amplification using a primer oligonucleotide set according to [1-17] in DNA genomic of a test plant in order to detect at least one polymorphic marker selected from the group consisting of PO1 to POS5; and, when the polymorphic marker is at least one element selected from the group consisting of PO1 to P03 and POS5, treat a PCR product obtained by PCR amplification with a restriction enzyme, and detect a product treated with a restriction enzyme .
[0040] [0040] [1-22] The method according to [1-21], in which the restriction enzyme is at least one element selected from the group consisting of Xbal, Kpnl, AfIII and Pvul.
[0041] [0041] [1-23] The method according to [1-21], wherein the polymorphic marker comprises P02 and PO5.
[0042] [0042] [1-24] The method according to [1-23], wherein the restriction enzyme comprises KpnlI and Pvul.
[0043] [0043] [1-25] The method according to any one of [1-21] to [1- 24], in which the plant obtained by the selection comprises 2% or more of rebaudioside M with respect to the amount of glycoside of total steviol contained in a leaf.
[0044] [0044] The present invention also provides the following.
[0045] [0045] [2-1] A non-genetically modified stevia plant with a high M rebaudioside content comprising 2% or more M rebaudioside in relation to the amount of total steviol glycoside contained in a dry leaf.
[0046] [0046] [2-2] The plant according to [2-1], additionally comprising 9.5% or more of rebaudioside D with respect to the amount of total steviol glycoside contained in a dry leaf.
[0047] [0047] [2-3] The plant according to [2-1] or [2-2], in which the plant is positive for at least one polymorphic marker selected from the group consisting of PO1, P02, PO3 , PO4 and POS5.
[0048] [0048] [2-4] A seed of the plant according to any of [2-1] to [2-3].
[0049] [0049] [2-5] A dry leaf of the plant according to any of [2-1] to [2-3].
[0050] [0050] [2-6] A tissue culture or plant cell grown from a plant according to any of [2-1] to [2-3].
[0051] [0051] [2-7] The tissue culture or the plant cell grown according to [2-6], where the tissue culture or the plant cell grown is an embryo, a meristematic cell, a pollen, a leaf, a root, a root apex, a petal, a protoplast, a leaf section or a callus.
[0052] [0052] [2-8] A method for producing a stevia plant with a high M rebaudioside content comprising 2% or more M rebaudioside with respect to the amount of total steviol glycoside contained in a dry leaf, the method comprising a step of crossing a stevia plant according to any one of [2-1] to [2-3] with a second stevia plant.
[0053] [0053] [2-9] The method according to [2-8], in which the second plant is the stevia plant according to any of [2-1] to [2-3].
[0054] [0054] [2-10] An extract of the plant according to any of [2- 1] to [2-3], the seed according to [2-4] or the dry leaf according to [2- 5).
[0055] [0055] [2-11] A food or drink product, a sweetening composition, a flavor or a medicine comprising the extract according to [2-10].
[0056] [0056] [2-12] A method for producing an extract containing rebaudioside M, comprising a step of obtaining an extract from the plant according to any one of [2-1] to [2-3], the seed of according to [2-4] or the dry leaf according to [2-5].
[0057] [0057] [2-13] A method for producing M rebaudioside, comprising a step of purifying M rebaudioside from an extract containing M rebaudioside according to [2-12].
[0058] [0058] [2-14] A method for producing a food or drink product, a sweetening composition, a flavor or a medicine, comprising a step of mixing an extract obtained by the method according to [2-12] and / or rebaudioside M obtained by the method according to [2-13] with other components.
[0059] [0059] [2-15] A method for selecting a stevia plant with a high M rebaudioside content, comprising a step of detecting at least one polymorphic marker selected from the group consisting of PO1, PO2, PO03, PO4 and POS from the genome of a test plant.
[0060] [0060] [2-16] The method according to [2-15], further comprising a step of measuring the content of rebaudioside M in a sheet tissue.
[0061] [0061] [2-17] Any one or more primer oligonucleotide set (s) selected from the group consisting of: (1) a primer oligonucleotide set comprising a primer oligonucleotide sense with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 2; (2) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 3 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 4; (3) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 5 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 6; (4) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 7 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 8; and (5) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 9 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 10, wherein the sequence of any 15 or more consecutive bases is positioned at the 3 'end of each primer oligonucleotide.
[0062] [0062] [2-18] A kit comprising at least one set of oligonucleotide primers selected from the sets of oligonucleotide primers (1) to (5) according to [2-17], and a restriction enzyme, in whereas the restriction enzyme is Xbal for the primer oligonucleotide set (1), the restriction enzyme is KpnIl for the primer oligonucleotide set (2), the restriction enzyme is AfIIl for the primer oligonucleotide set (3), and the restriction enzyme is Pvul for the oligonucleotide primer set (5).
[0063] [0063] [2-1] A probe comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 10, the probe being optionally linked with a detectable tag.
[0064] [0064] [2-20] The probe according to [2-19], in which the probe has a fluorescent label, a dye or a binding fraction.
[0065] [0065] [2-21] A method for selecting a genetically unmodified stevia plant with a high M rebaudioside content, comprising steps of: performing PCR amplification using a primer oligonucleotide set according to [2-17] in DNA genomics of a test plant in order to detect at least one polymorphic marker selected from the group consisting of PO1, PO02, PO03, PO4 and PO5; and, when the polymorphic marker is at least one element selected from the group consisting of PO1, P02, PO03 and POS, treat a PCR product obtained by PCR amplification with a restriction enzyme, and detect an enzyme-treated product restriction.
[0066] [0066] [2-22] The method according to [2-21], in which the restriction enzyme is at least one element selected from the group consisting of Xbal, Kpnl, AfIII and Pvul.
[0067] [0067] [2-23] The method according to [2-21], in which the polymorphic marker is PO02 and POS5.
[0068] [0068] [2-24] The method according to [2-23], wherein the restriction enzyme is KpnlI and Pvul.
[0069] [0069] [2-25] The method according to any one of [2-21] to [2- 24], in which the plant obtained by the selection comprises 2% or more of rebaudioside M with respect to the amount of glycoside of total steviol contained in a dry leaf. Advantageous effects of the invention
[0070] [0070] The present invention makes it possible to obtain a stevia plant rich in rebaudioside M and to provide an approach to produce a plant like this, a leaf obtained from a plant like this, and a food, drink, etc. ., containing rebaudioside M obtained from this sheet. [Brief description of the drawings]
[0071] [0071] [Figure 1] Figure 1 shows electrophoresis images obtained in the detection of the individual group marker [.
[0072] [0072] [Figure 2] Figure 2 shows electrophoresis images obtained in the detection of the individual group II marker. Description of Modalities
[0073] [0073] Hereinafter, the present invention will be described in detail. The modalities are provided below only to illustrate the present invention and are not intended to limit the present invention by such modalities. The present invention can be carried out in various ways without departing from the spirit of the present invention.
[0074] [0074] Please note that all documents, as well as public domain order publications, patent application publications, and other patent documents cited herein can be incorporated by reference. The present specification incorporates the contents of the specification and the drawings of the Japanese patent application 2017-198515, filed on October 12, 2017, from which the present application claims priority.
[0075] [0075] The present invention provides a genetically unmodified stevia plant with a high content of M rebaudioside comprising 2% or more of M rebaudioside with respect to the amount of total steviol glycoside contained in a leaf (for example, dry leaf or fresh leaf ) (hereinafter referred to as the “plant of the present invention” or “stevia plant of the present invention”).
[0076] [0076] The stevia plant of the present invention is a species derived from a stevia plant of wild species, but with a gene variation that increases the level of M rebaudioside. The gene variation occurs in a non-genetically modified manner (mentioned later).
[0077] [0077] In the present invention, among non-genetically modified stevia plants with a high M rebaudioside content, a stevia plant comprising 2 to 4.6% M rebaudioside with respect to the amount of total steviol glycoside contained in a leaf (for example example, dry leaf or fresh leaf), can also be referred to as a genetically unmodified stevia plant with a high M rebaudioside content, and a stevia plant comprising 4.7% or more M rebaudioside with respect to it can also be referred to as a stevia plant not genetically modified with ultra high content of rebaudioside M.
[0078] [0078] The total steviol glycoside (TSG) does not include an unknown steviol glycoside nor does it include a steviol glycoside present at a level below the detection limit. Preferably, the total steviol glycoside is any combination of two or more elements selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside 1, rebaudioside J, rebaudioside K, rebaudioside N, rebaudioside M, rebaudioside O, rebaudioside Q, rebaudioside R, dulcoside A, rubusoside, steviol, steviolmonoside, steviolbioside and stevioside. In a certain embodiment, the total steviol glycoside may consist of, for example, rebaudioside A, rebaudioside B, rebaudioside M, rebaudioside D, rebaudioside F, rebaudioside M and steviol. In another embodiment, the total steviol glycoside may consist of rebaudioside A, rebaudioside B, rebaudioside M, rebaudioside D, rebaudioside M, rebaudioside M, rebaudioside N, rebaudioside O and steviol.
[0079] [0079] The characteristic “comprising 2% or more of rebaudioside M with respect to the amount of total steviol glycoside contained in a leaf (for example, dry leaf or fresh leaf)” is distinguished in such a way that, when the rebaudioside content M (RebM) is indicated by RebM / TSG% as the ratio for the total amount of steviol glycosides obtained from a dry leaf, the lower limit of the RebM / TSG value is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, the previous characteristic is distinguished in such a way that the upper limit of the RebM / TSG value is 15% or less, 16% or less, 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. One with this ratio of 2% or more and less than 4.7% can be referred to as a phenotype with a high M rebaudioside content, and one with this ratio of 4.7% or more and 15% or less can be referred to as a phenotype with an ultra high content of rebaudioside M.
[0080] [0080] The plant of the present invention can also comprise 0.19 g or more of rebaudioside M in 100 g of a dry leaf. This means that when a dry leaf is obtained from the plant of the present invention, rebaudioside M is present in an amount of 0.19 g or more, 0.20 g or more, 0.25 g or more, 0.30 g or more, 0.35 g or more, 0.40 g or more, 0.45 g or more, 0.50 g or more, 0.55 g or more, 0.60 g or more, 0.65 g or more, 0.70 g or more, 0.75 g or more, 0.80 g or more, 0.85 g or more, 0.90 g or more, 0.95 g or more, 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1.25 g or more, 1.30 g or more, 1.35 g or more , 1.40 g or more, or 1.45 g or more per 100 g of dry leaf.
[0081] [0081] In this context, the dry leaf of the plant of the present invention refers to a leaf with a water content decreased by 3 to 4% by weight drying a fresh leaf of the stevia plant of the present invention.
[0082] [0082] In this context, the plant of the present invention may additionally comprise 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1 , 25 g or more, 1.30 g or more, 1.35 g or more, 1.40 g or more, 1.45 g or more, 1.50 g or more, 1.55 g or more, 1, 60 g or more, 1.65 g or more, 1.70 g or more, 1.75 g or more, 1.80 g or more, 1.85 g or more, 1.90 g or more, 1.95 g or more, 2.00 g or more, 2.05 g or more, 2.10 g or more, 2.15 g or more, 2.20 g or more, 2.25 g or more, 2.30 g or more, 2.35 g or more, 2.40 g or more, 2.45 g or more, 2.50 g or more, 2.55 g or more, 2.60 g or more, 2.65 g or more, 2.70 g or more, 2.75 g or more, 2.80 g or more, 2.85 g or more, 2.90 g or more, 2.95 g or more, 3.00 g or more , 3.05 g or more, 3.10 g or more, 3.15 g or more, 3.20 g or more, 3.25 g or more, 3.30 g or more, 3.35 g or more, 3.40 g or more, 3.45 g or more, 3.50 g or more, 3.55 g or more or 3.57 g or more of rebaudioside D in 100 g of a dry leaf.
[0083] [0083] In this context, the combination of the rebaudioside content M and D is not particularly limited and includes any combination.
[0084] [0084] Preferably, the plant of the present invention comprises 1.03 g or more of rebaudioside M and 1.1 g or more of rebaudioside D in 100 g of a dry leaf.
[0085] [0085] Alternatively, the leaf (e.g., dry leaf or fresh leaf) of the plant of the present invention, when the amount (g) of rebaudioside M contained per 100 g of a leaf of the wild type stevia plant is defined as 100% , can comprise higher rebaudioside M by 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 1100% or more, 1200% or more, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more, 2000% or more, 2100% or more, 2200% or more, 2300% or more, 2400% or more, 2500% or more, 2600% or more, 2700% or more, 2800% or more, 2900% or more, or 3000% or more compared to the species of wild type stevia.
[0086] [0086] The stevia plant of the present invention is distinguished in such a way that when the content of rebaudioside M (RebM) and rebaudioside D
[0087] [0087] The stevia plant of the present invention is distinguished in such a way that when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in a leaf (for example, dry leaf or fresh leaf) is indicated by (RebD + RebM) / TSG% as the ratio for the total amount of steviol glycosides, the lower limit of the (RebD + RebM) / TSG value is 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, it is distinguished in such a way that the upper limit of the (RebD + RebM) / TSG value is 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28 % or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. The ratio is preferably 14% or more and 40% or less, or 16% or more and 40% or less.
[0088] [0088] In an alternative embodiment, the plant of the present invention may have a total amount of steviol glycosides less than that of the wild type. Specifically, the plant of the present invention can comprise less than 19 g as the total amount of steviol glycosides in 100 g of a dry leaf. This means that when a dry leaf is obtained from the plant of the present invention, the total amount of steviol glycosides is less than 19 g, less than 18 g, less than 17 g, less than 16 g, less than 15 g, less than 14 g, less than 13 g, less than 12 g, less than 11 g, less than 10 g, less than 9 g, less than 8 g, or less than 7 g per 100 g of dry leaf.
[0089] [0089] The stevia plant of the present invention is distinguished in such a way that when the content of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of a leaf (for example, dry leaf or fresh leaf) is indicated for the RebM / RebA ratio, the lower limit of the RebM / RebA value is 0.03 or more, 0.04 or more, 0.05 or more, 0.06 or more, 0.08 or more, 0.10 or more, 0.12 or more, or 0.14 or more. On the other hand, it is distinguished in such a way that the upper limit of the RebM / RebA value is 0.08 or less, 0.10 or less, 0.12 or less, 0.14 or less, 0.16 or less, 0.18 or less, 0.20 or less, 0.24 or less, or 0.26 or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. The ratio is preferably 0.03 or more and 0.26 or less, or 0.10 or more and 0.26 or less.
[0090] [0090] The stevia plant of the present invention is distinguished in such a way that, when the content of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of a leaf (for example, dry leaf or fresh leaf) is indicated by (RebA + RebM) / TSG as the ratio for the total amount of steviol glycosides, the lower limit of the (RebA + RebM) / TSG value is 4% or more, 5% or more, 6% or more, 7 % or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24 % or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, it is distinguished in such a way that the upper limit of the value of (RebA +
[0091] [0091] As previously mentioned, the stevia plant of the present invention has a variation involved in increasing the level of rebaudioside M. Such variation has at least one of the following genomic characteristics (1) to (5) (hereinafter, can be referred to as the “genetic trait (s) of the present invention”).
[0092] [0092] (1) Homozygous for the allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T.
[0093] [0093] (2) Homozygous for the allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T.
[0094] [0094] (3) Homozygous for the allele in which the base in the position corresponding to position 41 of SEQ ID NO: 39 is C.
[0095] [0095] (4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
[0096] [0096] (5) Homozygous for the allele in which the base in the position corresponding to position 50 of SEQID NO: 43 is A.
[0097] [0097] The phrase "position (or portion) that corresponds to" means the following. If a sequence identical to a reference sequence (for example, SEQ ID NOs: 35, 37, 39, 42, 43, etc.) is present in the genome, it means a position or a portion in the sequence (for example , 44, 40, 41, 55-72, 50, etc.) present in the genome, and if a sequence identical to the reference sequence is not present in the genome, it means a position or portion in a sequence in the genome that corresponds to reference sequence, which corresponds to the position or portion in the reference sequence. Regardless of whether an identical sequence or which corresponds to the reference sequence exists or not in the genome, it can be determined, for example, by amplifying genomic DNA from the stevia plant of interest with a primer oligonucleotide capable of amplifying the reference sequence by PCR, sequencing the amplified product, and performing alignment analysis between the obtained sequence and the reference sequence. Non-limiting examples of a sequence that corresponds to a reference sequence include, for example, a base sequence with a sequence identity of 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 98.1% or more, 98.4% or more, 98.7% or more, 99.2% or more, 99.5% or more, or 99.8% or more with the reference sequence. The position or portion that corresponds to the position or portion in the reference sequence, in the sequence that corresponds to the reference sequence in the genome, can be determined by taking into account the base sequence before and after the position or portion in the reference sequence, and the like. For example, a position or portion in the sequence that corresponds to the reference sequence in the genome, which corresponds to a position or portion in the reference sequence, can be determined by an alignment analysis of a reference sequence, with a sequence that corresponds to a reference sequence in the genome.
[0098] [0098] For example, by taking “the position that corresponds to position 44 of SEQ ID NO: 35” of the genetic trait (1) as an example, in the case of the genome of a stevia plant presenting a portion consisting of a sequence base identical to SEQ ID NO: 35, “the position corresponding to position 44 of SEQ ID NO: 35" ”is position 44 from the 5 'end of the portion consisting of a base sequence identical to SEQ ID NO: 35. On the other hand, in the case that the genome of a stevia plant has a portion consisting of a base sequence which is not identical, but which corresponds to SEQ ID NO: 35, the genome does not have a portion consisting of a base sequence identical to SEQ ID NO: 35. Therefore, “the position that corresponds to position 44 of SEQ ID NO: 35" does not necessarily correspond to position 44 from the 5th end of the portion that corresponds to SEQ ID NO: 35 . However, it is possible to identify “the position that corresponds to position 44 of SEQ ID NO: 35” in the genome of a stevia plant like this, taking into account the base sequence before and after position 44 of SEQ ID NO: 35, and the like. For example, one can identify “the position that corresponds to position 44 of SEQ ID NO: 35” in the genome of a stevia plant by analyzing the base sequence alignment of a portion that corresponds to SEQ ID NO: 35 ” in the genome of a stevia plant, and the base sequence of SEQ ID NO: 35.
[0099] [0099] "The portion consisting of a base sequence corresponding to SEQ ID NO: 35" means, for example, a portion consisting of a base sequence with a sequence identity of 60% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.1% or more, 98.4% or more, 98.7% or more, 99% or more, 99.2% or more, 99.5% or more, or 99.8% or more with the base sequence of SEQ ID NO: 35.
[00100] [00100] In one embodiment, “the portion consisting of a base sequence that corresponds to SEQ ID NO: 35” includes a portion of the genome of a stevia plant that can be amplified by PCR using a sense primer, which hybridizes in a complementary sequence of a portion of 15 to 25 bases in size from the 5th end of SEQ ID NO: 35, and a reverse primer oligonucleotide that hybridizes in a portion of 15 to 25 bases in size from the 3rd end of SEQ ID NO: 35.
[00101] [00101] For simplicity, genetic trait (1) is used here as an example for explanation, but the same applies to genomic traits (2) to (5).
[00102] [00102] In a specific embodiment, "the portion consisting of a base sequence corresponding to SEQ ID NO: 35" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using an oligonucleotide sense primer comprising the base sequence of SEQ ID NO: 45, and an oligonucleotide reverse primer comprising the base sequence of SEQ ID NO0: 46.
[00103] [00103] In a specific embodiment, "the portion consisting of a base sequence corresponding to SEQ ID NO: 37" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using an oligonucleotide sense primer comprising the base sequence of SEQ ID NO47, and an oligonucleotide reverse primer comprising the base sequence of SEQ ID NO: 48.
[00104] [00104] In a specific embodiment, “the portion consisting of a base sequence corresponding to SEQ ID NO: 39” includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using an oligonucleotide sense primer comprising the base sequence of SEQ ID NO: 49, and a reverse primer comprising the base sequence of
[00105] [00105] In a specific embodiment, "the portion consisting of a base sequence corresponding to SEQ ID NO: 42" "includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using a sense primer oligonucleotide comprising the base sequence of SEQ ID NO: 51, and a reverse primer oligonucleotide comprising the base sequence of SEQ ID NO: 52.
[00106] [00106] In a specific embodiment, "the portion consisting of a base sequence corresponding to SEQ ID NO: 43" includes, for example, a portion of the genome of a stevia plant that can be amplified by PCR using an oligonucleotide sense primer comprising the base sequence of SEQ ID NO: 53, and a reverse primer comprising the base sequence of SEQ ID NO: 54.
[00107] [00107] In a specific embodiment, "the allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T" comprises the base sequence of SEQ ID NO: 55, 65 or 75.
[00108] [00108] In a specific embodiment, "the allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T" comprises the base sequence of SEQ ID NO: 57.67 or 77.
[00109] [00109] In a specific embodiment, "the allele in which the base in the position corresponding to position 41 of SEQ ID NO: 39 is C" comprises the base sequence of SEQ ID NO: 59, 69 or 79.
[00110] [00110] In a specific embodiment, "the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated" comprises the base sequence of SEQ ID NO: 61.71 or 81.
[00111] [00111] In a specific modality, "the allele in which the base in the position that corresponds to position 50 of SEQ ID NO: 43 is A" comprises the
[00112] [00112] Here, a position selected from the group consisting of (1) a position that corresponds to position 44 of SEQ ID NO: 35, (2) a position that corresponds to position 40 of SEQ ID NO: 37, (3) a position that corresponds to position 41 of SEQ ID NO: 39, (4) a portion that corresponds to positions 55-72 of SEQ ID NO: 42, and (5) a position that corresponds to position 50 of SEQ ID NO: 43 it can be referred to as a "polymorphic site of the present invention" or a "site of variation of the present invention".
[00113] [00113] Likewise, a selected variation from the group consisting of (1) a variation from A to T in a position that corresponds to position 44 of SEQ ID NO: 35, (2) a variation from C to T in a position that corresponds to position 40 of SEQ ID NO: 37, (3) a variation from G to C in a position that corresponds to position 41 of SEQ ID NO: 39, (4) an elimination of the portion that corresponds to to positions 55-72 of SEQ ID NO: 42, and (5) a variation from G to A in a position that corresponds to position 50 of SEQ ID NO: 43 can be referred to as a "polymorphism of the present invention" or a "variation of the present invention".
[00114] [00114] Previous genomic characteristics can be eliminated by the PCR method, TaqMan PCR method, sequencing method, microarray method, Invader method, TILLING method, RAD method (randomly amplified polymorphic DNA), fragment size polymorphism method restriction method (RFLP), PCR-SSCP method, AFLP method (amplified fragment size polymorphism), SSLP method (single sequence size polymorphism), CAPS method (cleaved amplified polymorphic sequence), dCAPS method (cleaved amplified polymorphic sequence) derivative), allele-specific oligonucleotide (ASO) method, ARMS method, gradient gel electrophoresis method
[00115] [00115] In a specific modality, the previous genomic characteristics are detectable on the basis of "polymorphic marker positivity" using a polymorphic marker developed by the present inventors.
[00116] [00116] In this context, the polymorphic marker is at least one element selected from the group consisting of PO1 to POS.
[00117] [00117] Positivity for PO1 means that only a band of approximately 383 bp in size (for example, SEQ ID NO: 21) is obtained: performing PCR amplification using a primer oligonucleotide felt with the nucleotide sequence shown in SEQ ID NO : 1, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 2 in the genomic DNA of a candidate plant; and treating the obtained PCR product (approximately 383 bp in size: for example, SEQ ID NO: 21 or 22) with an Xbal restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 344 bp (for example, SEQ ID NO: 23) is formed by treatment with Xbal restriction enzyme of the PCR product, the candidate plant is negative in relation to the POI.
[00118] [00118] Positivity in relation to PO02 means that only a band of approximately 297 bp in size (for example, SEQ ID NO:
[00119] [00119] Positivity in relation to PO3 means that only a band of approximately 390 bp in size (for example, SEQ ID NO: 27) is obtained: performing PCR amplification using a sense primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 5, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 6 in the genomic DNA of a candidate plant; and treating the obtained PCR product (approximately 390 bp in size) (for example, SEQ ID NO: 27 or 28) with an AfIII restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 347 bp (for example, SEQ ID NO: 29) is formed, the candidate plant is negative in relation to P03.
[00120] [00120] Positivity towards PO4 means that only a PCR product of approximately 140 bp (for example, SEQ ID NO: 30) is formed by performing PCR amplification using a primer oligonucleotide felt with the nucleotide sequence shown in SEQ ID NO: 7, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 8 in the genomic DNA of a candidate plant. When 140 bp (for example, SEQ ID NO: 30) and 158 bp (for example, SEQ ID NO: 34) PCR products are formed, the candidate plant is negative for the same.
[00121] [00121] Positivity towards POS means that only a band of approximately 288 bp in size (for example, SEQ ID NO: 31) is obtained: performing PCR amplification using a primer oligonucleotide felt with the nucleotide sequence shown in SEQ ID NO: 9, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 10 in the genomic DNA of a candidate plant; and treating the obtained PCR product (approximately 288 bp in size) (for example, SEQ ID NO: 31 or 32) with a Pvul restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 240 bp (for example, SEQ ID NO: 33) is formed, the candidate plant is negative in relation to PO5.
[00122] [00122] The term "approximately" in terms of the bp in size previously described means + 5 bp. Restriction enzyme treatment can be carried out according to conditions recommended by the distributor of each restriction enzyme used.
[00123] [00123] For the detailed method for selecting the plant of the present invention, see section “3. Method for selecting plant of the present invention ”mentioned later.
[00124] [00124] The previous genomic characteristics (for example, polymorphism) positive for the aforementioned polymorphic marker were confirmed in the examples because they present statistical correlation with phenotypes containing high M rebaudioside content and / or high D rebaudioside content.
[00125] [00125] The rebaudioside M or D can be extracted as a liquid extract by reacting a fresh leaf or a dry leaf of the plant of the present invention with a suitable solvent (an aqueous solvent such as water or an organic solvent such as an alcohol, ether or acetone). Regarding extraction conditions, etc., see a method described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3 (2010) or WO2010 / 038911, or a
[00126] [00126] The rebaudioside M can be further purified from the liquid extract thus obtained by using a method known in the art, such as a gradient of ethyl acetate or any of the other organic solvents: water, high performance liquid chromatography (HPLO ), gas chromatography, time-of-flight mass spectrometry (TOF-MS), or ultra (high) performance liquid chromatography (UPLC).
[00127] [00127] The content of rebaudioside M according to the present invention can be evaluated by a method described in Ohta et al., J. Appl. Glycosci., Vol. 57, No. 3 (2010) or WO2010 / 038911, or a method described in the examples mentioned later. Specifically, a fresh leaf can be sampled from the stevia plant of the present invention, followed by LC / MS-MS evaluation.
[00128] [00128] The plant of the present invention may include not only the complete plant, but a plant organ (for example, a leaf, a petal, a stem, a root, and a seed), a plant tissue (for example, epidermis, phloem, soft tissue, xylem, vascular bundle, palisade tissue, and spongy tissue), various forms of plant cells (e.g., cells grown in suspension), a protoplasty, a leaf section, a callus and the like. The leaf may be the dry leaf mentioned above.
[00129] [00129] The plant of the present invention can also include a tissue culture or a cultured plant cell. This is due to the fact that the plant can be regenerated by growing a tissue culture like this or a cultured plant cell. Examples of the tissue culture or cultured plant cell of the plant of the present invention include, but are not limited to, embryos, meristematic cells, pollens, leaves, roots, root apices, petals, protoplasts, leaf section and calluses.
[00130] [00130] In an alternative aspect, the present invention provides a method for producing a stevia plant with a high M rebaudioside content comprising 2% or more M rebaudioside with respect to the amount of total steviol glycoside contained in a leaf (for example , dry leaf or fresh leaf), the method comprising a step of crossing the stevia plant of the present invention with a second stevia plant (hereinafter, may be referred to as the "production method of the present invention").
[00131] [00131] The “stevia plant with a high content of M rebaudioside comprising 2% or more of M rebaudioside in relation to the amount of total steviol glycoside contained in a leaf” produced by the method has the same phenotype and genetic properties as those of the plant of present invention.
[00132] [00132] Specifically, when a leaf (for example, dry leaf or fresh leaf) is obtained from the plant obtained by the production method of the present invention, the leaf comprises 2% or more of rebaudioside M with respect to the amount of glycoside of total steviol contained in the leaf. This is distinguished in such a way that, when the rebaudioside M (RebM) content is indicated by RebM / TSG% as the ratio for the total amount of steviol glycosides obtained from the dry leaf, the lower limit of the RebM / TSG is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more. On the other hand, this means that the upper limit of the ReEbM / TSG value is 15% or less, 16% or less, 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28% or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. The ratio is preferably 2% or more and 20% or less, or 7% or more and 15% or less.
[00133] [00133] The plant obtained by the production method of the present invention can also comprise 0.19 g or more of rebaudioside M in 100 g of a dry leaf. This means that when a dry leaf is obtained from the plant of the present invention, rebaudioside M is present in an amount of 0.19 g or more, 0.20 g or more, 0.25 g or more, 0.30 g or more, 0.35 g or more, 0.40 g or more, 0.45 g or more, 0.50 g or more, 0.55 g or more, 0.60 g or more, 0.65 g or more, 0.70 g or more, 0.75 g or more, 0.80 g or more, 0.85 g or more, 0.90 g or more, 0.95 g or more, 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1.25 g or more, 1.30 g or more, 1.35 g or more , 1.40 g or more, or 1.45 g or more per 100 g of dry leaf.
[00134] [00134] In this context, the plant obtained by the production method of the present invention may additionally comprise 1.00 g or more, 1.05 g or more, 1.10 g or more, 1.15 g or more, 1.20 g or more, 1.25 g or more, 1.30 g or more, 1.35 g or more, 1.40 g or more, 1.45 g or more, 1.50 g or more, 1.55 g or more, 1.60 g or more, 1.65 g or more, 1.70 g or more, 1.75 g or more, 1.80 g or more, 1.85 g or more, 1.90 g or more, 1.95 g or more, 2.00 g or more, 2.05 g or more, 2.10 g or more, 2.15 g or more, 2.20 g or more, 2.25 g or more , 2.30 g or more, 2.35 g or more, 2.40 g or more, 2.45 g or more, 2.50 g or more, 2.55 g or more, 2.60 g or more, 2.65 g or more, 2.70 g or more, 2.75 g or more, 2.80 g or more, 2.85 g or more, 2.90 g or more, 2.95 g or more, 3 .00 g or more, 3.05 g or more, 3.10 g or more, 3.15 g or more, 3.20 g or more, 3.25 g or more, 3.30 g or more, 3, 35 g or more, 3.40 g or more, 3.45 g or more, 3.50 g or more, 3.55 g or more or 3.57 g or more of rebaudioside D in 100 g of Dry leaf.
[00135] [00135] In this context, the combination of rebaudioside content M and D is not particularly limited and includes any combination.
[00136] [00136] Preferably, the plant obtained by the production method of the present invention comprises 1.03 g or more of rebaudioside M and 1.1 g or more of rebaudioside D in 100 g of a dry leaf.
[00137] [00137] Alternatively, when the amount (g) of rebaudioside M contained per 100 g of a leaf (for example, dry leaf or fresh leaf) of the wild type stevia plant is defined as 100%, the leaf of the plant obtained by the method of production of the present invention may comprise rebaudioside M at a higher content of 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, 1100 % or more, 1200% or more, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900% or more, 2000% or more, 2100 % or more, 2200% or more, 2300% or more, 2400% or more, 2500% or more, 2600% or more, 2700% or more, 2800% or more, 2900% or more, or 3000% or more, compared to the wild type stevia species.
[00138] [00138] The plant obtained by the production method of the present invention is distinguished in such a way that when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in a leaf (for example, dry leaf or fresh leaf) is indicated for the RebM / RebD ratio, the lower limit of the RebM / RebD value is 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.8 or more, or 1.0 or more. On the other hand, it is distinguished in such a way that the upper limit of the RebM / RebD value is 0.3 or less, 0.4 or less, 0.5 or less, 0.6 or less, 0.8 or less, 1.0 or less, 1.1 or less, or 1.2 or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. The ratio is preferably 0.2 or more and 1.2 or less, or 0.6 or more and 1.1 or less.
[00139] [00139] The plant obtained by the production method of the present invention is distinguished in such a way that when the content of rebaudioside M (RebM) and rebaudioside D (RebD) in a leaf (for example, dry leaf or fresh leaf) is indicated by (RebD + RebM) / TSG% as the ratio for the total amount of steviol glycosides, the lower limit of the (RebD + RebM) / TSG value is 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, or 38% or more . On the other hand, it is distinguished in such a way that the upper limit of the (RebD + RebM) / TSG value is 18% or less, 20% or less, 22% or less, 24% or less, 26% or less, 28 % or less, 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, or 40% or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. The ratio is preferably 14% or more and 40% or less, or 16% or more and 40% or less.
[00140] [00140] In an alternative embodiment, the plant obtained by the production method of the present invention may have a total amount of steviol glycosides less than that of the wild type. Specifically, the plant obtained by the production method of the present invention can comprise less than 19 g as the total amount of steviol glycosides in 100 g of a dry leaf. This means that when a dry leaf is obtained from the plant of the present invention, the total amount of steviol glycosides per 100 g of the dry leaf is less than 19 g, less than 18 g, less than 17 g, less than 16 g, less than 15 g, less than 14 g, less than 13 g, less than 12 g, less than 11 g, less than 10 g, less than 9 g, less than 8 g, or less than 7 g.
[00141] [00141] The plant obtained by the production method of the present invention is distinguished in such a way that when the content of rebaudioside M (RebM) and rebaudioside A (RebA) in 100 g of a leaf (for example, dry leaf or fresh leaf ) is indicated by (RebA + RebM) / TSG as the ratio to the total amount of steviol glycosides, the lower limit of the (RebA + RebM) / TSG value is 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 14% or more, 16% or more, 18% or more, 20% or more, 22% or more, 24% or more, 26% or more, 28% or more, 30% or more, 32% or more, 34% or more, 36% or more, 38% or more, or 40% or more. On the other hand, it is distinguished in such a way that the upper limit of the (RebA + RebM) / TSG value is 30% or less, 32% or less, 34% or less, 36% or less, 38% or less, 40 % or less, 42% or less, 44% or less, 46% or less, 48% or less, 50% or less, 52% or less, 54% or less, 56% or less, 58% or less, 60 % or less, 62% or less, 64% or less, 66% or less, 68% or less, 70% or less, 72% or less, 74% or less, 76% or less, 78% or less, or 80% or less. The combination of the lower limit and the upper limit is not particularly limited, as long as the value of the upper limit is greater than the value of the lower limit in the combination. The ratio is preferably 4% or more and 80% or less, or 40% or more and 80% or less.
[00142] [00142] The plant obtained by the production method of the present invention has at least one of the following genomic characteristics (1) to (5) involved in increasing the level of rebaudioside M.
[00143] [00143] (1) Homozygous for the allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T.
[00144] [00144] (2) Homozygous for the allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T.
[00145] [00145] (3) Homozygous for the allele in which the base in the position corresponding to position 41 of SEQ ID NO: 39 is C.
[00146] [00146] (4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
[00147] [00147] (5) Homozygous for the allele in which the base in the position corresponding to position 50 of SEQID NO: 43 is A.
[00148] [00148] Such genomic characteristics are detectable as being "positive polymorphic marker", by the use of a polymorphic marker developed by the present inventors.
[00149] [00149] In this context, the polymorphic marker is at least one element selected from the group consisting of PO1 to PO5.
[00150] [00150] Positivity to PO1l means that only a band of approximately 383 bp in size (eg SEQ ID NO: 21) is obtained: performing PCR amplification using a sense primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 1, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 2 in the genomic DNA of a candidate plant; and treating the obtained PCR product (approximately 383 bp in size: for example, SEQ ID NO: 21 or 22) with an Xbal restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 344 bp (for example, SEQ ID NO: 23) is formed by treatment with restriction enzyme Xbal of the PCR product, the candidate plant is negative in relation to PO1.
[00151] [00151] Positivity to PO02 means that only a band of approximately 297 bp in size (for example, SEQ ID NO: 24) is obtained: performing PCR amplification using a sense primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 3, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 4 in the genomic DNA of a candidate plant; and treating the obtained PCR product (297 bp in size) (for example, SEQ ID NO: 24 or 25) with a KpnI restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 258 bp (for example, SEQ ID NO: 26) is formed, the candidate plant is negative in relation to P02.
[00152] [00152] Positivity in relation to PO03 means that only a band of approximately 390 bp in size (for example, SEQ ID NO: 27) is obtained: performing PCR amplification using a primer oligonucleotide felt with the nucleotide sequence shown in SEQ ID NO: 5, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 6 in the genomic DNA of a candidate plant; and treating the obtained PCR product (approximately 390 bp in size) (for example, SEQ ID NO: 27 or 28) with an AfIII restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 347 bp (for example, SEQ ID NO: 29) is formed, the candidate plant is negative in relation to P03.
[00153] [00153] Positivity towards PO4 means that only a PCR product of approximately 140 bp (for example, SEQ ID NO: 30) is formed by performing PCR amplification using a primer oligonucleotide felt with the nucleotide sequence shown in SEQ ID NO: 7, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 8 in the genomic DNA of a candidate plant. When 140 bp (for example, SEQ ID NO: 30) and 158 bp (for example, SEQ ID NO: 34) PCR products are formed, the candidate plant is negative in relation to it.
[00154] [00154] Positivity to PO5 means that only a band of approximately 288 bp in size (for example, SEQ ID NO: 31) is obtained: performing PCR amplification using a sense primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 9, and a reverse primer oligonucleotide with the nucleotide sequence shown in SEQ ID NO: 10 in the genomic DNA of a candidate plant; and treating the obtained PCR product (approximately 288 bp in size) (for example, SEQ ID NO: 31 or 32) with a Pvul restriction enzyme. On the other hand, when a product treated with a restriction enzyme of approximately 240 bp (for example, SEQ ID NO: 33) is formed, the candidate plant is negative in relation to POS5.
[00155] [00155] The term "approximately" in relation to bp in size previously described means +5 bp. Restriction enzyme treatment can be carried out according to conditions recommended by the distributor of each restriction enzyme used.
[00156] [00156] In the production method of the present invention, "hybridize" means that the plant of the present invention (first generation (S1)) is crossed with a second plant (S1) to obtain a young plant of the same (plant produced by the method of production of the present invention (second generation (82))). The method of hybridization is preferably backcross. “Backcrossing” is an additional crossover approach from a young plant (S2), generated between the plant of the present invention and the second plant, with the plant of the present invention (ie, a plant with the characteristic (s) ) genetics (s) of the present invention) (S1) to produce a plant with the genetic characteristic (s) of the present invention. When the second plant (S1) for use in the production method of the present invention has the same phenotype and genetic properties as those of the plant of the present invention, the crossing is substantially backcross. The genetic polymorphism of the present invention can be inherited according to Mendel's law. In association with this, the phenotype correlates with the genetic polymorphism, that is, the phenotype with a high M rebaudioside content can also be inherited according to Mendel's law.
[00157] [00157] Alternatively, the plant of the present invention can also be produced by self-fertilization. Self-fertilization can be accomplished by self-pollinating the pollen of the plant stamen of the present invention with the plant pistil of the present invention.
[00158] [00158] Since the plant produced by the production method of the present invention has the same phenotype and genetic properties as the plant of the present invention, the plant produced by the production method of the present invention can be further crossed with a third stevia plant to produce a stevia plant with a high M rebaudioside content comprising 2% or more M rebaudioside, with respect to the amount of total steviol glycoside contained in a leaf (for example, dry leaf or fresh leaf).
[00159] [00159] In an alternative embodiment, the plant of the present invention can be produced by regenerating a plant by cultivating the tissue culture or cultured plant cell mentioned above. The cultivation conditions are the same as for cultivating a tissue culture or a cultured plant cell of the wild type stevia plant, and are known in the art (Protocols for In Vitro cultures and secondary metabolite analysis of aromatic and medicinal plants, Method in molecular biology, vo. 1391, pp. 113-123).
[00160] [00160] Alternatively, the plant of the present invention is in a non-genetically modified form and, therefore, can also be prepared by integrating the polymorphism previously described in the wild type stevia plant by a non-genetic modification approach. Examples of the "non-genetic modification approach" include a method of inducing a variation in the gene of a host cell (or a host plant) without transfection with a foreign gene. Examples of a method like this include a method of allowing a mutagen to act in a plant cell. Examples of a mutagen such as this include ethyl methanesulfonate (EMS) and sodium azide. For example, ethyl methanesulfonate (EMS) can be used in a concentration such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7 %, 0.8%, 0.9%, or 1.0% to treat a plant cell. The treatment time is 1 to 48 hours, 2 to 36 hours, 3 to 30 hours, 4 to 28 hours, 5 to 26 hours, or 6 to 24 hours. The treatment procedures themselves are known in the art and can be carried out by immersing an absorbed seed in water, obtained through a water absorption process, in a treatment solution containing the mutagen at the concentration described above for the treatment time previously described.
[00161] [00161] An alternative example of the non-genetic modification approach may be a method of irradiating a plant cell with radiation or a beam of light such as X-rays, y-rays, or ultraviolet rays. In this case, an irradiated cell that uses an appropriate dose (ultraviolet lamp intensity, distance and time) of ultraviolet ray is grown in a selective or similar medium, and then a cell, a callus, or a plant with the characteristic of interest can be selected. In this use, the irradiation intensity is 0.01 to 100 Gr, 0.03 to 75 Gr, 0.05 to 50 Gr, 0.07 to 25 Gr, 0.09 to 20 Gr, 0.1 to 15 Gr, 0.1 to 10 Gr, 0.5 to 10 Gr, or 1a 10 Gr. The irradiation distance is 1 cm to 200 m, 5 cm to 100 m, 7 cm to 75 m, 9 cm to 50 m, 10 cm at 30 m, 10 cm at 20 m, or 10 cm at 10 m. The irradiation time is 1 minute to 2 years, 2 minutes to 1 year, 3 minutes to 0.5 year, 4 minutes to 1 month, minutes to 2 weeks, or 10 minutes to 1 week. The irradiation intensity, distance and time differ depending on the type of radiation or the state of the subject to be irradiated (cell, callus or plant), and can be adjusted appropriately by those skilled in the art.
[00162] [00162] Approaches such as cell fusion, anther culture (haploid reproduction), and remote crossing (haploid reproduction) are also known in the art.
[00163] [00163] In general, plant cells can involve a mutation during cultivation. Therefore, it is preferable to regenerate an individual plant, to maintain the characteristic more stably.
[00164] [00164] Although the plant of the present invention is a stevia plant not genetically modified, the scope of the present invention does not exclude a plant obtained by the ex-post facto recombination gene (eg genome editing) with the plant of the present invention as a host (for example, a plant additionally provided with another
[00165] [00165] The plant of the present invention or the plant with the same phenotype and genetic properties, as those of the plant of the present invention, can be selected by detecting the genetic characteristic (s) of the present invention from a tissue of this plant. In this context, "selecting" means that the plant of the present invention is discriminated from other plants to select the plant of the present invention.
[00166] [00166] Thus, in an alternative aspect, the present invention provides a method for selecting a stevia plant with a high content of M rebaudioside, comprising a step of detecting the presence and / or absence of at least one of the following genomic characteristics ( 1) to (5), the following genomic characteristics, from the genome of a test plant (hereinafter, may be referred to as the "method of selection of the present invention").
[00167] [00167] (1) Homozygous for the allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T.
[00168] [00168] (2) Homozygous for the allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T.
[00169] [00169] (3) Homozygous for the allele in which the base in the position corresponding to position 41 of SEQ ID NO: 39 is C.
[00170] [00170] (4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
[00171] [00171] (5) Homozygous for the allele in which the base in the position corresponding to position 50 of SEQID NO: 43 is A.
[00172] [00172] The method for selecting the present invention may further comprise a step of selecting among the test plants a plant, in which the presence of at least one genetic characteristic of
[00173] [00173] The presence of the genetic characteristic (s) of the present invention can be determined: by detecting the presence of an allele selected from the group consisting of: (A) an allele in which the base in the position corresponding to position 44 of SEQ ID NO: 35 is T;] (B) an allele in which the base in the position corresponding to position 40 of SEQ ID NO: 37 is T; (C) an allele in which the base at the position corresponding to position 41 of SEQID NO: 39 is C; (D) an allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated; and (E) an allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is A; and / or detecting the absence of an allele selected from the group consisting of: (A) an allele in which the base at the position corresponding to position 44 of SEQ ID NO: 35 is A; (b) an allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is C; (c) an allele in which the base at the position corresponding to position 41 of SEQ ID NO0: 39 is G; (d) an allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is not eliminated; and (e) an allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is G.
[00174] [00174] The absence of the genetic characteristic (s) of the present invention can be determined:
[00175] [00175] Specific examples of methods of detecting the genomic characteristics of the present invention include, but are not limited to, PCR method, TaqMan PCR method, sequencing method, microarray method, Invader method, TILLING method, RAD method, RFLP method, PCR-SSCP method, AFLP method, SSLP method, CAPS method,
[00176] [00176] In the case of the PCR method, it is preferable to generate an oligonucleotide primer in such a way that the portion of the 3rd end has a sequence complementary to the polymorphic site of the present invention. Using a primer oligonucleotide designed in this way, the polymerase extension reaction continues, as a result of the primer oligonucleotide completely hybridizing in the mold, if the mold sample shows the polymorphism, whereas if the mold does not show the variation of the present invention, the extension reaction does not occur, because the nucleotide at the 3rd end of the initiator oligonucleotide is incompatible with the template. Therefore, PCR amplification is performed using an oligonucleotide primer like this, and the amplification product is analyzed by agarose gel electrophoresis or similar, and if an amplification product of a predetermined size can be confirmed, the mold as the sample shows a variation, and if the amplification product is not present, it can be judged that the mold does not show a variation.
[00177] [00177] Alternatively, the genetic characteristic (s) of the present invention - can be detected by designing the primer oligonucleotide sequence in such a way that the polymorphism of the present invention and the oligonucleotide sequence primers do not overlap, and the gene variation of the present invention can be amplified by PCR, and by sequencing the base sequence of the amplified nucleotide fragment.
[00178] [00178] Regarding PCR and agarose gel electrophoresis see
[00179] [00179] The TaqMan PCR method uses fluorescent labeled allele-specific oligonucleotides and Taq DNA polymerases (Livak, K. J. Genet). Anal. 14, 143 (1999); Morris T. et al., J. Clin. Microbiol. 34, 2933 (1996)).
[00180] [00180] The sequencing method is a method for analyzing the presence or absence of a variation by amplifying a region containing the variation by PCR, and sequencing the DNA sequence using a dye or similar finisher (Sambrook, Fritsch and Maniatis, “Molecular Cloning : A Laboratory Manual ”2nd Edition (1989), Cold Spring Harbor Laboratory Press).
[00181] [00181] A DNA microarray is one in which one end of a nucleotide probe is immobilized in an arrangement on a support, and includes a DNA chip, a Gene chip, a microchip, a sphere array and the like. Using a probe containing a sequence complementary to the polymorphism of the present invention, the presence or absence of the polymorphism of the present invention can be comprehensively detected. DNA microarray assays, such as DNA chips, include GeneChip assays (see Affymetrix; U.S. patents 6,045,996;
[00182] [00182] The invasive method combines the hybridization of two reporter probes specific to each allele of a polymorphism, such as SNPs and an invasive probe, in template DNA and the cleavage of DNA by the enzyme Cleavase with a special endonuclease activity, which cleaves a DNA by recognizing its structure (Livak, KJ Biomol. Eng. 14, 143-149 (1999); Morris T. et al., J. Clin. Microbiol. 34, 2933 (1996); Lyamichev, V. et al. , Science, 260, 778-783 (1993), and the like).
[00183] [00183] The TILLING method (Local lesions induced by bleaching in genomes) is a method in which the mutational incompatibility in the genomes of a mutated mutant population is selected by PCR amplification and treatment with CEL I nuclease.
[00184] [00184] In one embodiment, the selection method of the present invention may comprise a step of detecting polymorphic marker positivity from the genome of a test plant. A step like this comprises an operation of extracting genomic DNA from the test plant, and detecting the polymorphic marker of the present invention from genomic DNA. The polymorphic marker is, as already mentioned, at least one element selected from the group consisting of PO1 to PO5. Polymorphic marker positivity means that it is positive for at least one marker selected from the group consisting of PO1 to POS.
[00185] [00185] The method of detecting positivity in relation to PO1 to POS is as already mentioned.
[00186] [00186] In this embodiment, the selection method of the present invention can further comprise a step of selecting a plant in which the positive polymorphic marker of the present invention is detected among the test plants.
[00187] [00187] In a certain embodiment, the present invention provides a method for selecting a genetically unmodified stevia plant with a high content of M rebaudioside, comprising steps of: performing PCR amplification using at least one of the following sets of the primer oligonucleotide (1 ) to (5) in genomic DNA from a test plant; and treating a PCR product obtained by PCR amplification with a restriction enzyme, and detecting a restriction enzyme treated product: (1) a set of the primer oligonucleotide comprising
[00188] [00188] The conditions for PCR amplification are as already mentioned.
[00189] [00189] However, the primer oligonucleotide set is not limited to those with the sequences of SEQ ID NOs: 1 to 10. For example, the sense primer oligonucleotide can present at its 3rd end a sequence from the 3rd end of SEQ ID NO : 1,3,5,7 or 9 to 15 bases upstream of it (see the table below), and the reverse primer oligonucleotide can present at its 3rd end a sequence from the 3 'end of SEQ ID NO: 2 , 4, 6, 8 or 10 to 15 bases upstream of it (see the table below). Such a primer oligonucleotide can have 15 to 50 bases in size or 20 to 45 bases in size. Table 1 polymorphic marker a | Primer oligonucleotide sense Reverse primer oligonucleotide to be detected (sequence from the 3rd end | (sequence from the 3 'end at 15 bases upstream) PO1 (1st) IS -ATTTATTGTATCTAG-3' (SEQ ID) 5 "-GTACACATGCTACAC-3 '(SEQ NO: 11) ID NO: 12) PO2 (2') 5" -CAAACAACCGGGTAC-3 '(SEQ | 5 "-AGACATTGGCAACTC-3' (SEQ 1D NO: 13 ) ID NO: 14) PO3 (3) S5 "-ACGAAACCCGCTTAA-3 '(SEQ | 5" -TAATCCTTGAATTAG-3' (SEQ 1D NO: 15) ID NO: 16) PO4 5 "-ACACGTATACTAATC-3 '(SEQ | 5 "-CATGGTATGTACAAC-3 '(SEQ a) 1D NO: 17) ID NO: 18) PO5 S" -CATTCATGAGCGATC-3' (SEQ | 5 "-AAATCCCATATGTAG-3 '(SEQ 5º 1D NO: 19. ID NO: 20)
[00190] [00190] The primer oligonucleotide set is not limited to those with the sequences of SEQ ID NOs: 1 to 10. For example, the sense primer oligonucleotide can present or comprise a sequence of any 15 or more consecutive bases in SEQ ID NO: 1 , 3, 5, 7 or 9, and the reverse primer may have or comprise a sequence of any 15 or more consecutive bases in SEQ ID NO: 2,4,6,8 or 10.
[00191] [00191] (17) A primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 1 and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more bases consecutive in SEQ ID NO: 2; (2) a primer oligonucleotide set comprising a primer oligonucleotide felt with or
[00192] [00192] An oligonucleotide primer like this can be 15 to 50 bases in size, 20 to 45 bases in size, or 30 to 65 bases in size, as long as the arbitrary sequence of 15 or more consecutive bases is present at the 3 'end.
[00193] [00193] The selection methods of the present invention may further comprise a step of determining the RebM content of a tissue (e.g., a leaf) from the tissue of the test stevia plant, for the
[00194] [00194] (1) Detecting the genetic characteristic (s) of the present invention from the genome of a stevia test plant; (11) determine the RebM content of the tissue of the test stevia plants, in which the genetic trait (s) is / are detected; (iii) selecting an individual with a higher RebM content among the stevia test plants, in which the genetic characteristic (s) of the present invention is / are detected; (iv) crossing the selected individual with a higher RebM content with another stevia plant; (v) detecting the genetic characteristic (s) of the present invention from the genome of daughter plants obtained by crossing, (vi) assessing the RebM content of the tissue of the daughter plants, in which the (s) genetic trait (s) is / are detected, (vii) select individuals with a higher RebM content among the daughter plants, in which genomic characteristics are detected.
[00195] [00195] Individuals with a high RebM content of choice can be, for example, up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, up to 4%, up to 3% , up to 2%, or up to 1% of test stevia plants, in which the genetic characteristic (s) of the present invention is / are detected, with respect to the high content of RebM. Other stevia plants to be crossed may or may not contain the genetic characteristic (s) of the present invention. In the previous embodiment, steps (iv) to (vile) can be repeated a plurality of times. In this way, stevia plants with a higher RebM content can be selected.
[00196] [00196] In the selection method of the present invention, the test stevia plant can be a natural plant or a non-transgenic plant. Non-transgenic plants are as described in the plant-related section of the present invention.
[00197] [00197] In the screening method of the present invention, the test stevia plant may include a stevia plant subjected to a mutagenesis treatment and a plant of the progeny thereof. Mutagenesis treatment is as described in the plant-related section of the present invention, and includes treatment with a mutagen, treatment with radiation or irradiation with light, and the like.
[00198] [00198] The present invention also provides the primer oligonucleotide set mentioned above, for example, any one or more primer oligonucleotide set (s) selected from the group consisting of the primer oligonucleotide sets (1) to ( 5), (11) to (57) and (17) to (5 ") above. The present invention further provides a primer oligonucleotide set capable of amplifying a region with a base sequence selected from the group consisting of SEQ ID NOs: 35 to 44 by PCR, for example, a sense primer oligonucleotide comprising a SEQ base sequence ID NO: 45, a primer oligonucleotide set with a reverse primer oligonucleotide comprising a base sequence of SEQ ID NO: 46, a sense primer comprising a base sequence of SEQ ID NO: 47, a set of the primer oligonucleotide with a primer reverse primer oligonucleotide comprising a base sequence of SEQ ID NO: 48, a sense primer oligonucleotide comprising a base sequence of SEQ ID NO: 49, a set of the primer oligonucleotide with a reverse primer oligonucleotide comprising a base sequence of SEQ ID NO: : 50, a set of the primer oligonucleotide with a sense primer oligonucleotide comprising a base sequence of SEQ ID NO: 51, a set of the primer oligonucleotide with a reverse primer comprising a base sequence of SEQ ID NO: 52, a sense primer comprising a base sequence of SEQ ID NO: 53, and a reverse set of the primer oligonucleotide with a base sequence of SEQ ID NO: 54.
[00199] [00199] Furthermore, the present invention provides a probe capable of detecting the presence and / or absence of the genomic characteristics of the present invention, which can be referred to as the "probe of the present invention" hereinafter. The probe of the present invention can have a structure suitable for various methods of detection for the presence and / or absence of the genetic characteristic (s) of the present invention. For example, the probe of the present invention may comprise a base sequence complementary to a portion of a genome comprising a variation site of the present invention. Non-limiting examples of such probes include those comprising a base sequence selected from SEQ ID NOs: 55 to 64. Of these sequences, SEQ ID NOs: 55, 57, 59, 61 and 63 are specific to alleles comprising the variation of the present invention, and SEQ ID NOs: 56, 58, 60, 62 and 64 are specific for alleles that do not contain the variation of the present invention. The presence of the genetic characteristic (s) of the present invention can be detected by detecting an allele comprising the variation (s) of the present invention, and / or by not detecting an allele that does not comprise the variation (s) of the present invention, and the absence (s) of the genetic characteristic (s) of the invention by not detecting an allele comprising the variation (s) of the present invention , and / or by detecting an allele that does not comprise the variation (s) of the present invention. The probes of the present invention preferably have a label. Non-limiting examples of such labels include fluorescent labels, luminescent labels, radioactive labels, dyes, enzymes, finalizers, binding fractions with detectable labels and the like. In a specific embodiment, the probe of the present invention has a base sequence selected from SEQ ID NOs: 55 to 64 and a label.
[00200] [00200] The present invention further provides a Kkit for selection comprising any one or more primer oligonucleotide set (s) selected from the group consisting of the primer oligonucleotide sets (1) to (5), 11) a (57) and (1) to (5), and optionally a restriction enzyme.
[00201] [00201] In the kit, the restriction enzyme contained in the kit is Xbal in the case of using any one or more set (s) of the starter oligonucleotide selected (s) from the group, which consists of the sets of the starter oligonucleotide (1), (1) and (17).
[00202] [00202] In the kit, the restriction enzyme contained in the kit is Kpnl, in the case of using any one or more set (s) of the primer oligonucleotide selected (s) from the group consisting of the sets of the primer oligonucleotide (2), (2nd) and (2).
[00203] [00203] In the kit, the restriction enzyme contained in the kit is AfIII, in the case of using any one or more set (s) of the primer oligonucleotide selected (s) from the group consisting of the sets of the primer oligonucleotide (3), (3rd) and 3 ”).
[00204] [00204] In the kit, the restriction enzyme contained in the kit is Pvul, in the case of using any one or more set (s) of the primer oligonucleotide selected (s) from the group consisting of the sets of the primer oligonucleotide (5), (57) and (5).
[00205] [00205] In another embodiment of the kit: in the case of the primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 contiguous bases or more in SEQ ID NO: 1, the restriction enzyme comprises Xbal; in the case that the primer oligonucleotide pool comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 3, the restriction enzyme comprises Kpnl; in the case that the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 5, the restriction enzyme comprises AfIII; and in the case that the primer oligonucleotide pool comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 9, the restriction enzyme comprises Pvul.
[00206] [00206] The present invention also provides a selection kit comprising a set of the primer oligonucleotide capable of PCR amplifying a region with a base sequence selected from the group consisting of SEQ ID NOs: 35 to 44, and a probe of the present invention .
[00207] These sets of the primer oligonucleotide, probes and kits can be used to detect the genetic characteristic (s) of the present invention, used in the selection methods of the present invention and the like. These primer oligonucleotide sets and kits may also comprise an instruction that includes an explanation in detecting the genetic trait (s) of the present invention, and in the method of selecting the present invention, for example, a written instruction and means of communication, for example, a floppy disk, a CD, a DVD, a Blu-ray disk, a memory card, a USB memory, etc., with information recorded on them regarding the method of use.
[00208] [00208] In a further aspect, the present invention provides a method for producing an extract containing rebaudioside M, comprising a step of obtaining an extract from the plant of the present invention, or a seed or a leaf (for example, dry leaf or fresh leaf) of the plant (hereinafter, may be referred to as the "extract production method of the present invention"). The present invention further provides a method for producing rebaudioside M, comprising a step of purifying rebaudioside M from an extract obtained by the extract production method of the present invention (hereinafter, it may be referred to as the “rebaudioside M method of production of the present invention ”).
[00209] [00209] Specifically, the present invention provides a method for producing rebaudioside M or rebaudioside D, or both, comprising a step of obtaining an extract containing rebaudioside M or rebaudioside D, or both, from the stevia plant with a high content of rebaudioside M of the present invention, the stevia plant with a high M rebaudioside content selected by the selection method of the present invention, or the stevia plant with a high M rebaudioside content produced by the method of the present invention.
[00210] [00210] The extract containing rebaudioside M or rebaudioside D, or both, can be obtained by reacting a fresh leaf or a dry leaf of the plant of the present invention with a suitable solvent (an aqueous solvent such as water or an organic solvent such as an alcohol , ether or acetone). For extraction conditions, etc., see a method described in Ohta et al., J.
[00211] [00211] Rebaudioside M or rebaudioside D, or both, can be purified from the extract containing rebaudioside M or rebaudioside D, or both by using a method known in the art, such as an ethyl acetate gradient or any of the other organic solvents: water, high performance liquid chromatography (HPLC), gas chromatography, time-of-flight mass spectrometry (TOF-MS), or ultra (high) performance liquid chromatography (UPLC).
[00212] [00212] The extract obtained by the extract production method of the present invention (hereinafter, may be referred to as the "extract of the present invention") comprises rebaudioside M or rebaudioside D, or both, in higher content compared to the species of wild type stevia.
[00213] [00213] The extract of the present invention may comprise rebaudioside M or rebaudioside D, or both, in a higher content of 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800 % or more, 900% or more, 1100% or more, 1200% or more, 1300% or more, 1400% or more, 1500% or more, 1600% or more, 1700% or more, 1800% or more, 1900 % or more, 2000% or more, 2100% or more, 2200% or more, 2300% or more, 2400% or more, 2500% or more, 2600% or more, 2700% or more, 2800% or more, 2900 % or more, 3000% or more, 3100% or more, 3200% or more, 3300% or more, 3400% or more, 3500% or more, 3600% or more, 3700% or more, 3800% or more, 3900 % or more, 4000% or more, 4100% or more, 4200% or more, 4300% or more, 4400% or more, 4500% or more, 4600% or more, 4700% or more, 4800% or more, 4900 % or more, or 5000% or more compared to an extract obtained from the wild type stevia species. The extract of the present invention and the extract obtained from the wild type stevia species can be that obtained by the same
[00214] [00214] The extract of the present invention thus obtained and / or rebaudioside M or rebaudioside D, or both, obtained by the method to produce rebaudioside M or rebaudioside D, or both, according to the present invention, can be mixed with other components for produce an unprecedented drug, flavor or food or drink product with a higher content of rebaudioside M or rebaudioside D, or both. Accordingly, in an alternative aspect, the present invention provides a method for producing a medicament, flavor or food or drink product, comprising a step of mixing the extract of the present invention and / or rebaudioside M or rebaudioside D, or both , obtained by the method to produce rebaudioside M or rebaudioside D, or both, according to the present invention with other components. The present invention additionally provides a drug, flavor or novel food or drink product with a higher content of rebaudioside M or rebaudioside D, or both, obtained by the production method. In this context, the food or drink product means a drink and a food. Thus, in a certain embodiment, the present invention provides an unprecedented drug, flavor, drink or food and also provides a method for producing the drug, flavor, drink or food. [Examples]
[00215] [00215] Hereinafter, the present invention will be described with reference to experimental examples, examples, etc. However, the present invention is not limited by these specific modalities. [Example 1] Preparation of stevia plant with high RebM content
[00216] [00216] Commercially available stevia seeds were sown, increased in August 2014, and about 3,000 individuals were subjected to selection based on development and growth condition and leaf morphology during the period from October 2014 to March 2015. The content and content ratio of each steviol glycoside of 115 individuals selected in April 2015 were assessed by quantitative analysis using LC-MS / MS, and 3 individuals each with a high ratio of RebD and RebM (in each individual, the ratio of the total content of the two components to the content of total steviol glycoside (TSG) was 20% or more) were selected. At the same time, selection based on total steviol glycoside (TSG) content was also performed, and 14 individuals with a TSG yield (the total amount of steviol glycosides evaluated per dry leaf weight unit) of 20% or more were selected.
[00217] [00217] From the 115 individuals previously selected, 5 individuals with high content of the RebA type showing each good development were selected. Artificial inter-individual or intra-individual pollination started in January 2015, and mass seed production was carried out in March 2015 to obtain the first self-fertilized generation (S1 seeds). In addition, S1 seeds were sown and increased, and S1 populations of 105 individuals were obtained. In June 2015, each individual of these S1 populations was grown separately to obtain self-fertilized second generations (S2 seeds) by artificial intra-individual pollination. The resulting S2 seeds were distinguished between the respective S1 individuals as strains to obtain S2 populations of 105 strains. - Subsequently, the resulting S2 individuals were investigated for the condition of development and growth, and four leaves were sampled from each individual that exhibits good development and growth were subjected to quantitative analysis of the amount of steviol glycosides using LC-MS / MS. The RebD content, RebM content and TSG content were calculated from the analytical results obtained. An individual with the sum of the RebD content and the RebM content per 100 g of leaves that is greater than 2 g (= 2%) was selected as an excellent individual.
[00218] [00218] Plants with a high content of RebD and RebM (3 individuals) and plants with a high content of TSG (6 individuals) selected by component analysis were subjected, as parent plants for reproduction, in a crossing test involving 53 combinations in total. The selected individuals were propagated vegetatively by cutting in April 2015 to establish seedlings in June 2015, which were then matured until November 2015 to establish a plurality of parental stocks for each individual. The artificial crossing started in January 2016, and about 1,000 hybrid seeds (F1 seeds) for each combination were obtained. Subsequently, sowing and increasing of the resulting F1 seeds started in March 2015. Subsequently, the resulting F1 individuals were investigated for the condition of development and growth, and four leaves were sampled from each individual that exhibits good development and growth and were subjected to quantitative analysis of the amount of steviol glycosides using LC-MS / MS. The RebD content, RebM content and TSG content were calculated from the analytical results obtained. An individual in which the sum of the RebD content and the RebM content was greater than 2 g / 100 g (= 2%) was selected as an excellent individual.
[00219] [00219] Genetic markers were prepared using 10 individuals obtained from the initial selection. Individuals were classified in June 2015 into 2 groups, that is, a high-content group (29.16% or more) and a low-content group (6.06% or less), based on the sum of the contents of RebD and RebM. Research regarding the common polymorphism in each
[00220] [00220] For the detection of the PO1 marker, PCR was performed using the primer oligonucleotides provided below. A restriction enzyme (Xbal) was added to the PCR product, and the enzymatic reaction was carried out at 37ºC for treatment with the restriction enzyme. After treatment with restriction enzyme, electrophoresis was performed using a LabChip GX Touch HT microchip type electrophoresis device. The marker was identified on the basis of a band pattern after electrophoresis.
[00221] [00221] The primer oligonucleotide sequences are as follows.
[00222] [00222] Fw primer oligonucleotide: 5- AAGGTTCTTTATTTTTAAACTTATGTTAATTTATTGTATCTAG-3 '(SEQ ID NO: 1) Rv primer oligonucleotide: 5 * - CCTTATGTACACATGCTACAC-3' (SEQ ID::
[00223] [00223] When a product treated with a restriction enzyme of approximately 344 bp (for example, SEQ ID NO: 23) was not formed by treatment with Xbal restriction enzyme of the obtained PCR product (approximately 383 bp in size), the test in question was listed as being positive for PO1. (2) PO2
[00224] [00224] For the detection of the PO02 marker, PCR was performed using the
[00225] [00225] The primer oligonucleotide sequences are as follows.
[00226] [00226] Fw primer oligonucleotide: 5- TAATCATCCAAACCCTAATCTCGCCAAACAACCGGGTAC-3 '- (SEQ ID NO: 3) Rv primer oligonucleotide: 5- GAGGAAGACATTGGCAACTC-3' (SEQ ID NO: 4)
[00227] [00227] When a product treated with a restriction enzyme of approximately 260 bp (for example, SEQ ID NO: 26) was not formed by treatment with the restriction enzyme KpnI of the obtained PCR product (approximately 297 bp in size), the test in question was listed as being positive for P02. (3) PO03
[00228] [00228] For the detection of the P03 marker, PCR was performed using the primer oligonucleotides provided below. A restriction enzyme (AfIII) was added to the PCR product, and the enzymatic reaction was carried out at 37ºC for treatment with the restriction enzyme. After treatment with restriction enzyme, electrophoresis was performed using a LabChip GX Touch HT microchip type electrophoresis device. The marker was identified on the basis of a band pattern after electrophoresis.
[00229] [00229] The primer oligonucleotide sequences are as follows.
[00230] [00230] Oligonucleotide Fw primer: 5-
[00231] [00231] When a product treated with a restriction enzyme of approximately 347 bp (eg SEQ ID NO: 29) was not formed by treatment with restriction enzyme AfIII of the obtained PCR product (approximately 390 bp in size), the test in question was listed as being positive for P03. (4) PO4
[00232] [00232] For the detection of the PO4 marker, PCR was performed using the primer oligonucleotides provided below. Electrophoresis was performed on the PCR product using a LabChip GX Touch HT microchip electrophoresis device. The marker was identified on the basis of a band pattern after electrophoresis.
[00233] [00233] The primer oligonucleotide sequences are as follows.
[00234] [00234] Fw primer oligonucleotide: 5- CGCAAACACGTATACTAATC-3 '(SEQ ID NO: 7) Rv primer oligonucleotide: 5 * - TTTAGCATGGTATGTACAAC-3' (SEQ ID NO: 8)
[00235] [00235] When only a PCR product of approximately 140 bp (for example, SEQ ID NO: 30) was formed, the test in question was listed as being positive for PO04. (5) POS
[00236] [00236] For the detection of the POS marker, PCR was performed using the primer oligonucleotides provided below. A restriction enzyme (PvulD) was added to the PCR product, and the enzymatic reaction was carried out at 37ºC for treatment with the restriction enzyme. After treatment with restriction enzyme, electrophoresis was performed using a LabChip GX Touch HT microchip type electrophoresis device. The marker was identified on the basis of a band pattern after electrophoresis.
[00237] [00237] The primer oligonucleotide sequences are as follows.
[00238] [00238] Oligonucleotide Fw primer: 5-
[00239] [00239] When a product treated with a restriction enzyme of approximately 240 bp (for example, SEQ ID NO: 33) was not formed by treatment with Pvul restriction enzyme of the obtained PCR product (approximately 288 bp in size), the test in question was listed as being positive for PO5.
[00240] [00240] A marker verification experiment was performed using the polymorphic markers previously established and individual group 1. Component analysis was performed on 192 individuals from the individual group [, and 8 individuals from the top (0.35% or more) and 8 individuals from the fund (0.12% or less) were selected on the basis of RebM content values, followed by the marker test described previously. As a result, only the top 8 individuals with a high RebM content were selected as positive individuals for the polymorphism of interest (Figure 1).
[00241] [00241] The verification test was also performed in the individual group TT in the same way as before. Component analysis was performed on 137 individuals from individual group II, and 8 individuals from the top (0.24% or more) and 8 individuals from the bottom (0.01% or less) were selected on the basis of RebM content values , followed by the marker test. As a result, only the top 8 individuals with a high RebM content were selected as individuals with the polymorphism of interest (Figure 2). As is evident from figures 1 and 2, the band with the size of interest appeared only in individuals with a phenotype with a high RebM content.
[00242] [00242] An experiment was carried out for additional verification using the increased number of individuals in the two segregating populations used to verify the genetic markers, that is, individual groups I and II. 62 individuals and 109 individuals, respectively, including the numbers of individuals described above were used. Each individual group was divided on the basis of the RebM content into 3 groups: 0.2% or more, 0.1% or more, less than 0.2%, and 0% or more, less than 0.1% . As a result of performing the marker test, the group of 0.2% or more was preferably detected with the marker of the present invention. These results demonstrate that the frequency of appearance of positive individuals was statistically significant between the groups (fit quality test using the chi square test; the null hypothesis established that the frequency distribution occurred even without the association of the marker test results with the phenotype; for the test results, see the tables below). Table 2 Marker test results in individual group I (62 individuals) * Marker used: P02 Chi-square test result (df = 2) 36.81 ** Table 3 Marker test results in individual group I (62 individuals)
[00243] [00243] As a result of selecting a plant with a high RebM content using the genetic markers obtained previously, individuals with a RebM ratio of 2% or more were also able to be selected from segregating populations other than the populations for verification, shown in the table below, confirming that these genetic markers are capable of functioning as markers of practical selection. The results of selecting a plant with a high RebM content are shown in the table below. In the table, the circle mark represents that the marker test results were positive.
[00244] [00244] The present invention enables the most efficient provision of rebaudioside M and rebaudioside D and, therefore, can provide a drug, a flavor or a food or drink product, etc. comprising sufficient amounts of rebaudioside M and rebaudioside D and thereby having good taste quality.

权利要求:
Claims (25)
[1]
1. Stevia plant not genetically modified with high M rebaudioside content, characterized by the fact that it comprises 2% or more M rebaudioside in relation to the amount of total steviol glycoside contained in a leaf.
[2]
2. Plant according to claim 1, characterized by the fact that it additionally comprises 9.5% or more of rebaudioside D in relation to the amount of total steviol glycoside contained in a leaf.
[3]
3. Plant according to claim | or 2, characterized by the fact that it has at least one of the following genomic characteristics (1) to (5).
(1) Homozygous for the allele in which the base at the position corresponding to position 44 of SEQ ID NO: 35 is T.
(2) Homozygous for the allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is T.
(3) Homozygous for the allele in which the base at the position corresponding to position 41 of SEQ ID NO: 39 is C.
(4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
(5) Homozygous for the allele in which the base at the position corresponding to position 50 of SEQID NO: 43 is A.
[4]
4. Plant according to any of the claims | to 3, characterized by the fact that the plant is positive for at least one polymorphic marker selected from the group consisting of PO1 to PO5.
[5]
5. Seed, tissue, tissue culture or cultured plant cell, characterized by the fact that it is from the plant as defined in any of the claims | to 4.
[6]
6. Tissue, tissue culture or the plant cell grown according to claim 5, characterized (a) by the fact that it is an embryo, a meristematic cell, a pollen, a leaf, a root, a root apex, a petal, protoplasty, leaf section or callus.
[7]
7. Method for producing a stevia plant with a high M rebaudioside content, characterized by the fact that it comprises 2% or more M rebaudioside in relation to the amount of total steviol glycoside contained in a leaf, the method comprising a crossing step a stevia plant as defined in any of the claims | to 4, with a second stevia plant.
[8]
Method according to claim 7, characterized in that the second plant is the stevia plant as defined in any one of claims 1 to 4.
[9]
9. Plant extract as defined in any of the claims | to 4, characterized by the fact that it is from the seed, tissue, tissue culture or cell as defined (o) in claim 5.
[10]
10. Food or drink product, a sweetening composition, a flavor or a medicine, characterized (a) by the fact that it comprises the extract as defined in claim 9.
[11]
11. Method for producing an extract containing rebaudioside M, characterized in that it comprises a step of obtaining an extract from the plant as defined in any one of claims 1 to 4, from the seed, tissue, tissue culture or cell as defined (o) in the claim
5.
[12]
12. Method for producing M rebaudioside, characterized in that it comprises a step of purifying M rebaudioside from an extract containing M rebaudioside as defined in claim 11.
[13]
13. Method for producing a food or drink product, a sweetening composition, a flavor or a medicine, characterized by the fact that it comprises a step of mixing an extract obtained by the method as defined in claim 12 and / or rebaudioside M obtained by the method as defined in claim 13 with other components.
[14]
14. Method for selecting a stevia plant with a high content of M rebaudioside, characterized by the fact that it comprises a step of detecting the presence and / or absence of at least one of the following genomic characteristics (1) to (5) from the genome of a test plant.
(1) Homozygous for the allele in which the base at the position corresponding to position 44 of SEQ ID NO: 35 is T.
(2) Homozygous for the allele in which the base at the position corresponding to position 40 of SEQ ID NO: 37 is T.
(3) Homozygous for the allele in which the base at the position corresponding to position 41 of SEQ ID NO: 39 is C.
(4) Homozygous for the allele in which the portion corresponding to positions 55-72 of SEQ ID NO: 42 is eliminated.
(5) Homozygous for the allele in which the base at the position corresponding to position 50 of SEQ ID NO: 43 is a.
[15]
Method according to claim 14, characterized in that it comprises a step of detecting at least one polymorphic marker selected from the group consisting of PO1 to PO5 from the genome of a test plant.
[16]
16. Method according to claim 14 or 15, characterized in that it additionally comprises a step of measuring the content of rebaudioside M in a sheet tissue.
[17]
17. Any one or more primer oligonucleotide set (s) selected from the group, characterized by the fact that it consists of: (1) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 1, and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 2; (2) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 3, and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 4; (3) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 5, and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NC: 6; (4) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 7, and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 8; and (5) a primer oligonucleotide set comprising a primer oligonucleotide felt with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 9, and a reverse primer oligonucleotide with or comprising a sequence of any 15 or more consecutive bases in SEQ ID NO: 10, where the sequence of any 15 or more consecutive bases is positioned at the 3rd end of each primer oligonucleotide.
[18]
18. Kit, characterized in that it comprises a primer oligonucleotide set as defined in claim 17, and optionally a restriction enzyme, wherein in the case of the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 contiguous bases or more in SEQ ID NO: 1, the restriction enzyme comprises Xbal; in the case that the primer oligonucleotide pool comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 3, the restriction enzyme comprises Kpnl; in the case that the primer oligonucleotide set comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 5, the restriction enzyme comprises AfIII; and in the case that the primer oligonucleotide pool comprises a primer oligonucleotide felt with or comprising a sequence of any 15 consecutive bases or more in SEQ ID NO: 9, the restriction enzyme comprises Pvul.
[19]
19. Probe comprising a nucleotide sequence shown in any of SEQ ID NOs: 55 to 64, characterized by the fact that the probe is optionally linked with a detectable tag.
[20]
20. Probe according to claim 19, characterized in that the probe has a fluorescent label, a dye or a binding fraction.
[21]
21. Method for selecting a genetically unmodified stevia plant with a high M rebaudioside content, characterized by the fact that it comprises steps of: performing PCR amplification using a set of the primer oligonucleotide as defined in claim 17, in genomic DNA of a plant test with the purpose of detecting at least one polymorphic marker selected from the group consisting of PO1 to POS; and, when the polymorphic marker is at least one element selected from the group consisting of PO1 to PO03 and POS,
treating a PCR product obtained by PCR amplification with a restriction enzyme, and detecting a product treated with restriction enzyme.
[22]
22. Method according to claim 21, characterized by the fact that the restriction enzyme is at least one element selected from the group consisting of Xbal, KpnlI, AfITI and Pvul.
[23]
23. Method according to claim 21, characterized by the fact that the polymorphic marker is PO2 and POS5.
[24]
24. The method of claim 23, characterized by the fact that the restriction enzyme is KpnI and Pvul.
[25]
25. Method according to any of claims 21 to 24, characterized in that the plant obtained by the selection comprises 2% or more of rebaudioside M with respect to the amount of total steviol glycoside contained in a leaf.
Figure 1
INDIVIDUAL WITH HIGH INDIVIDUAL WITH LOW REB CONTENT; REB CONTENT »3 86 o To ooo Ss 3 EE TM sv AS vo Ae vTr wins qo 7TT7 E nm
3. -) PO1 SO rm ms AS a ss pa OS OS CON a O = no- '2 Ex; "” PO2 00 -—— in— a —— - ——s— = E i E PO03 E mr - EN - a POA [1 Mn e - = -. E.: ã PO5 060 ln Im Mn Mn E Mon is Jus Es e iu te 200 Figure 2 INDIVIDUAL WITH HIGH: INDIVIDUAL WITH LOW CONTENT OF REB: CONTENT OF REB SIN ToOoNÃSom; muymuUoYoN No mv TO T7 MT! Ty Tvs 7 & | Mm Os MO 7 7 o PO1 SO rr MU E ca —— MM to AA ao - To aa O 207: - so 1 Zoo Po2 200 - am - - - as—. Ss ss .— - s— s— ss —ss— >>) E. À e> a> a E] E, uu aaa PO3 |. == === PO4 ——n ———————- = - = - = - Fo: 00 E. e) PO5 300 - at MO A ra pa a Aa a ana aa oo

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法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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JP2017-198515|2017-10-12|

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JP2017198515|2017-10-12|

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PCT/JP2018/038064|WO2019074089A1|2017-10-12|2018-10-12|Rebaudioside m-rich stevia plant|

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