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BR-122026002301-A2 - MODIFIED TOBACCO PLANT, RECOMBINANT DNA CONSTRUCTOR, CURED TOBACCO MATERIAL, TOBACCO PRODUCT, INVENTION AND METHODS FOR IMPROVING LEAF QUALITY IN A TOBACCO PLANT, FOR PRODUCING A TOBACCO PRODUCT AND FOR PRODUCING THE MODIFIED TOBACCO PLANT

BR122026002301A2BR 122026002301 A2BR122026002301 A2BR 122026002301A2BR-122026002301-A2

Abstract

The present disclosure includes methods and compositions for increasing leaf quality in low-alkaloid tobacco plants, for example, by combining inducible promoters and non-coding RNAs for suppression of an ornithine decarboxylase (ODC) gene. Also provided are low-alkaloid tobacco plants with normal, suppressed, or otherwise altered levels of polyamine. Also provided are tobacco plants with altered total alkaloid, nicotine levels, commercially acceptable leaf grade, their development through breeding or transgenic approaches, and the production of tobacco products from these tobacco plants.

Inventors

  • MARCOS FERNANDO DE GODOY LUSSO
  • JAMES A. STRICKLAND
  • Jesse Frederick
  • Dongmei Xu
  • Chengalrayan Kudithipudi

Assignees

  • ALTRIA CLIENT SERVICES LLC

Dates

Publication Date
20260310
Application Date
20190304
Priority Date
20180305

Claims (13)

  1. 1. A modified low-alkaloid tobacco plant, characterized by comprising a recombinant nucleic acid molecule comprising an inducible promoter operationally linked to a transcribable DNA sequence encoding a non-coding RNA for the deletion of an ornithine decarboxylase (ODC) gene.
  2. 2. Modified low-alkaloid tobacco plant according to claim 1, characterized in that said ODC gene comprises a nucleotide sequence that has at least 90% identity to a sequence selected from the group consisting of SEQ IDs 23, 24, 25, 26, 27 and 28.
  3. 3. Modified low-alkaloid tobacco plant according to claim 1, characterized in that said inducible promoter comprises a sequence selected from the group consisting of the SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21.
  4. 4. Modified low-alkaloid tobacco plant, characterized by comprising a recombinant nucleic acid molecule comprising a spike-inducible promoter operationally linked to a transcribable DNA sequence encoding a non-coding RNA for the deletion of an ornithine decarboxylase (ODC) gene, said gene having at least 90% identity with a sequence selected from the group consisting of SEQ IDs 23, 24, 25, 26, 27 and 28.
  5. 5. Modified low-alkaloid tobacco plant, characterized by comprising a nícl nic2 genotype and further comprising a recombinant nucleic acid molecule comprising a spike-inducible promoter operationally linked to a transcribable DNA sequence encoding a non-coding RNA for the deletion of an ornithine decarboxylase (ODC) gene having at least 90% identity with a sequence selected from the group consisting of SEQ IDs NOs: 23, 24, 25, 26, 27 and 28.
  6. 6. Modified low-alkaloid tobacco plant according to any one of claims 1 to 5, characterized in that said modified low-alkaloid tobacco plant is capable of producing a leaf comprising a level of one or more polyamines within at least 20% relative to a comparable leaf of a control tobacco plant, a chlorophyll level within at least 20% relative to a comparable leaf of a control tobacco plant, a number of mesophyll cells per unit area of leaf within at least 20% relative to a comparable leaf of a control tobacco plant, an epidermal cell size within at least 20% relative to a comparable leaf of a control tobacco plant, a leaf yield within at least 20% relative to a comparable leaf of a control tobacco plant, or any combination thereof; wherein said control tobacco plant does not comprise said recombinant nucleic acid molecule.
  7. 7. Recombinant nucleic acid molecule, characterized in that it comprises an inducible promoter operationally linked to a transcribable DNA sequence encoding a non-coding RNA for the deletion of an ornithine decarboxylase (ODC) gene.
  8. 8. Recombinant nucleic acid molecule, characterized in that it comprises an operationally spike-inducible promoter linked to a transcribable DNA sequence encoding a non-coding RNA for the deletion of an ornithine decarboxylase (ODC) gene, having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25, 26, 27 and 28.
  9. 9. Cured tobacco material characterized by being from the low-alkaloid tobacco plant modified as defined in any one of claims 1 to 6.
  10. 10. Tobacco product characterized by comprising cured tobacco material as defined in claim 9.
  11. 11. A method for improving leaf quality in a low-alkaloid tobacco plant, characterized in that it comprises: a. cultivating a low-alkaloid tobacco plant; b. suppressing the expression or activity of an ornithine decarboxylase (ODC) gene in said tobacco plant to create a low-alkaloid tobacco plant; and c. harvesting leaves from said modified low-alkaloid tobacco plant.
  12. 12. A method for producing a tobacco product, characterized in that it comprises: a. obtaining cured tobacco material as defined in claim 9; and b. producing a tobacco product from said cured tobacco material.
  13. 13. Method for producing modified low-alkaloid tobacco plant, as defined in any one of claims 1 to 6, characterized in that it comprises: a) obtaining said recombinant nucleic acid molecule; b) introducing said recombinant nucleic acid molecule into a low-alkaloid tobacco plant or a part of a low-alkaloid tobacco plant; and c) selecting a modified low-alkaloid tobacco plant or part of a modified low-alkaloid tobacco plant from step b) comprising said recombinant nucleic acid molecule.

Description

CROSS-REFERENCE WITH RELATED REQUEST [001]This request claims priority to U.S. Provisional Application No. 62/638,928, filed March 5, 2018, and is incorporated by reference in this descriptive report in its entirety. INCORPORATION OF SEQUENCE LISTING [002]A sequence listing contained in the ASCII file named “P34584W000_SL.txt” which is 88,011 bytes in size (measured in MS-Windows®) and was created on March 4, 2019, is electronically deposited herewith and incorporated by reference in its entirety. FIELD [003]The present disclosure includes tobacco plants having altered total alkaloid and nicotine levels and commercially acceptable leaf grade, their development through breeding or transgenic approaches, and the production of tobacco products from these tobacco plants. FUNDAMENTALS [004]Tobacco is one of the most widely grown non-food crops in the world, with global production exceeding 7.4 million tons (FAOSTAT, “Food and Agriculture Organization of the United Nations” (FAO) (2014), faostat.fao.org) and resulting tobacco products having an annual global market size of US$770 billion (“Euromonitor International”, 2016). Nicotine is the main alkaloid that accumulates in tobacco leaves. Nicotine and other minor alkaloids are also precursors to tobacco-specific nitrosamines (TSNA). There are demands for the development of tobacco cultivars with lower nicotine levels. [005]In commercial tobacco cultivars, nicotine represents 90-95% of the total alkaloid pool or 2-5% of the total dry weight of the leaf (Saitoh F., Nona M., Kawashima N. (1985). “The Alkaloid Contents of Sixty Nicotiana Species”. Phytochem. 24: 477-480). Nicotine is synthesized in the roots (Dawson RF (1942) “Accumulation of Nicotine in Reciprocal Grafts of Tomato and Tobacco”. Am. J. Bot. 29: 66-71), and translocalized through the xylem (Baldwin I.T. (1988). “The Alkaloidal of Wild Tobacco to Real and Simulated Herbivory”. Oecologia 77: 378-381) to the aerial parts of the plant (Hildreth S.B., Gehman E.A., Yang H., Lu R.H., Ritesh K.C., Harich K.C., Yu S., Lin J., Sandoe J.L., Okumto S., Murphyd, A.S., Jeleskoaet J.G. (2011). “Tobacco Nicotine Uptake Permease (NUP1) Affects Alkaloid Metabolism.” Proc. Natl. Acad. Sci. USA 108: 18.179-18.184) where it accumulates in the leaves and is exuded by trichomes in response to insect herbivory (Kessler A., Baldwin I.T. (2002). “Plant Responses to Insect Herbivory: the Emerging Molecular Analysis”. Annu. Rev. Plant Biol. 53: 299-328). Nicotine biosynthesis is influenced by genetic factors, plant development, biotic and abiotic stresses, phytohormonal signals, and agronomic management practices such as shoot apex and regrowth (Wang S.S., Shi Q.M., Li W.Q., Niu J.F., Li C.J., Zhang F.S. (2008). “Nicotine Concentration in Leaves of Flue-Cured Tobacco Plants as Affected by Removal of the Shoot Apex and Lateral Buds”. J. Integr. Plant Bio. 50: 958-964; Shoji T., Hashimoto T. (2015). “Stress-Induced Expression of NICOTINE2- Locus Genes and Their Homologs Encoding Ethylene Response Factor Transcription Factors in Tobacco”. Phytochem. 113: 41-49). The genetic regulation of nicotine biosynthesis correlates with two independent loci, Nic1 and Nic2, which have a synergistic effect on nicotine levels, but the effect of Nic1 is approximately 2.4 times stronger than that of Nic2 (Legg P.D., Collins G.B. (1971). “Inheritance of Percent Total Alkaloids in Nicotiana tabacum L. II. Genetic Effects of Two Loci in Burley 21 x LA Burley 21 Populations”. Can. J. Genet. Cytol. 13: 287-291). Both loci also influence the expression of several other genes unrelated to the nicotine biosynthesis pathway (Kidd S.K., Melillo A.A., Lu R.H., Reed D.G., Kuno N., Uchida K., Furuya M., Jelesko J.G. (2006). “The A and B loci in Tobacco Regulate a Network of Stress Response Genes, Few of Which Are Associated With Nicotine Biosynthesis”. Plant Mol. Biol. 60: 699-716; Shoji T., Kajikawa M., Hashimoto T. (2010). “Clustered Transcription Factor Genes Regulate Nicotine Biosynthesis in Tobacco”. Plant Cell 22: 3390-3409). Transcriptional analysis demonstrated that the Nic2 locus is a gene cluster that encodes at least seven ethylene response transcription factors (ERFs) (Shoji et al. 2010). [006]Homozygous mutations of one or both loci can be used to create nearly isogenic Burley 21 lines with reduced alkaloid levels, i.e., a high-intermediate (HI) variety with the nic2 genotype, a low-intermediate (LI) variety with the nic1 genotype, and an alkaloid-poor (LA) variety with the nic1nic2 genotype (Legg P.D., Chaplin J.F., Collins G.B. (1969). “Inheritance of Percent Total Alkaloids in Nicotiana tabacum L”. J. Hered. 60: 213-217; Legg et al. 1971). Burley 21 LA plants contain only approximately 5.7% of the total alkaloid levels found in the wild-type variety with normal alkaloid (NA) content (Legg P.D., Collins G.B., Littion C.C. (1970). “Registration of LA Burley 21 Tobacco Germplasm”. Crop. Sci. 10: 212). In LA plants, the synergistic effect of nic1 an