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US-12622375-B2 - High rebaudioside M stevia plant cultivars and methods of producing the same

US12622375B2US 12622375 B2US12622375 B2US 12622375B2US-12622375-B2

Abstract

Stevia varieties with a high content of RebM, are disclosed Further provided are methods for producing Stevia plants having a high RebM content by negatively regulating certain genes selecting the resulting plants, and breeding with such plants to confer such desirable Reb M phenotypes to plant progeny.

Inventors

  • Avetik Markosyan
  • Seong Siang Ong
  • Runchun Jing
  • Tengfang Huang
  • Fayaz Khazi
  • Stephen Ezra Schauer
  • Indra Prakash
  • Alec Hayes

Assignees

  • PURECIRCLE USA INC.
  • KEYGENE N.V.
  • THE COCA-COLA COMPANY

Dates

Publication Date
20260512
Application Date
20231213

Claims (5)

  1. 1 . A Stevia rebaudiana plant comprising at least one disrupted negative regulator gene in the rebaudioside A to rebaudioside M conversion pathway, and wherein the negative regulator gene is selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.
  2. 2 . A method of increasing the rebaudioside M content in a Stevia rebaudiana plant by inducing a disruption in at least one negative regulator gene function in the plant, wherein said negative regulator gene is selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.
  3. 3 . A plant part of the Stevia rebaudiana plant of claim 1 , wherein the plant part is selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyl, meristematic cells, ovules, seeds, cells, roots, root tips, pistils, anthers, flowers, and stems.
  4. 4 . A plant, plant part, or cell produced by the method of claim 2 , comprising in its genome or more genes affecting metabolism and selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10.
  5. 5 . A method of producing a Stevia plant comprising the steps of: a) introducing a recombinant DNA construct comprising a nucleotide sequence comprising one or more sequences selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10 operably linked to at least one heterologous nucleotide sequence into a Stevia rebaudiana plant, wherein said heterologous nucleotide sequence is a promoter; b) growing a fertile, mature plant resulting from step a); and c) selecting a plant expressing the at least one heterologous nucleotide sequence in at least one plant tissue.

Description

CROSS REFERENCE TO RELATED APPLICATION This application is a divisional application which claims the benefit of priority to U.S. application Ser. No. 17/701,154, now U.S. Pat. No. 11,844,323 issue date of Dec. 19, 2023 which is a divisional of U.S. application Ser. No. 16/491,470, now U.S. Pat. No. 11,284,577 issued Mar. 29, 2022 which is a 371 National Stage entry of and claims the benefit of priority to PCT Application No. PCT/US2018/021389 filed on Mar. 7, 2018, which claims the benefit of priority to U.S. Provisional Application No. 62/468,937, filed on Mar. 8, 2017, the entire contents of which are incorporated herein by reference for all purposes. SUBMISSION OF SEQUENCE LISTING The Sequence Listing associated with this application is filed in electronic format via PatentCenter under the xml file PURC08USG1 Sequence Listing and is hereby incorporated by reference into the specification in its entirety. BACKGROUND Stevia is an important and valuable field crop for the production of sweeteners, sugar substitutes, and other consumable ingredients. Thus, a continuing goal of stevia plant breeders is to develop stable, high yielding stevia cultivars of stevia species that are agronomically sound. The reasons for this goal are to maximize the amount and quality of the sweeteners, sugar substitutes, and other consumable ingredients. To accomplish this goal, the stevia breeder must select and develop plants that have the traits that result in superior cultivars. All references cited herein are incorporated by reference in their entirety. The development of new stevia cultivars requires the evaluation and selection of parents and the crossing of these parents. The lack of predictable success of a given cross requires that a breeder, in any given year, make several crosses with the same or different breeding objectives. The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification. SUMMARY The following embodiments and aspects thereof are described and illustrated in conjunction with products and methods, which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements. It is believed that the conversion of rebaudioside A to rebaudioside M in the plant requires minimal steps to accomplish. However, known Stevia species contain very little rebaudioside M compared to rebaudioside A, suggesting, that the conversion pathway is subject to negative effect of certain regulating elements in these plants. By identifying these negative regulators and disrupting their functionality, it is believed that the conversion pathway from rebaudioside A to rebaudioside M can occur more readily, resulting in a higher yield of rebaudioside M in the Stevia leaf An embodiment of the present disclosure is directed to producing Stevia rebaudiana plant cultivars which produce high levels of rebaudioside M as compared to native or commercially known Stevia plants such as Stevia rebaudiana Bertoni and Stevia rebaudiana Morita. Another embodiment discloses the disruption of negative regulators of rebaudioside M biosynthesis. By disrupting the function of these negative regulators in Stevia, Stevia plants can be produced that are able to produce higher levels of rebaudioside M as compared to different Stevia varieties. Another embodiment discloses a Stevia rebaudiana plant comprising at least one disrupted negative regulator gene in the rebaudioside A to rebaudioside M conversion pathway. Another embodiment discloses a method of increasing the rebaudioside M content in a Stevia rebaudiana plant by inducing a disruption in at least one negative regulator gene function in the plant, wherein said negative regulator gene is selected from the group consisting of genes affecting metabolism, signal transduction and gene regulation, and other novel uncategorized genes. Another embodiment discloses metabolism genes, wherein said genes are comprised of genes affecting sugar metabolism, mono-oxygenase content, terpene metabolism, aminotransferase metabolism, multi-antimicrobial extrusion protein metabolism, and methionine metabolism. Another embodiment discloses metabolism genes, wherein said genes are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10. Another embodiment discloses a plant, or a plant part thereof, of the Stevia rebaudiana plant of the present application, consisting of leaves, pollen, embryos, cotyledons, hypocotyl, meristematic cells, ovules, seeds, cells, roots, root tips, pistils, anthers, flowers, and stems. Another embodiment discloses signal transduction and gene re