Transgenic plant, methods and compositions for producing the same

Abstract

The invention relates to biotechnology and provides novel engineered protein for conferring enhanced growth. The invention also provides transgenic plants, seeds, cells and plant parts containing recombinant DNA molecules, as well as methods of using the same. In some embodiments, the invention relates to recombinant DNA molecules encoding enzymes that confer demethylase activity of RNA.

Description

Transgenic Plant, Methods and Compositions for Producing the SameBackground of the Invention(i) Field of the Invention

[0001] This disclosure relates to the compositions and methods for improving plant development and stress tolerance in plants.(ii) Sequence Listing

[0002] Tire Sequence Listing in an ASCII text file, named 00020_00001_WO_Seq.txt of 51,761 bytes, created on February 24, 2026. and submitted to the United States Patent and Trademark Office via EFS-Web, is incorporated herein by reference.(iii) Description of Related Art

[0003] To nourish an increasing population both physically and mentally, developing plant varieties that are more productive and tolerant to stress is a critical goal of plant scientists. Plant biotechnology has played a significant role in increasing crop production (Das et al., Selvaraj et al.. Various transgenes have been used to increase crop production (U.S. Pat. Nos. 12,145,967 and 11,046,969).

[0004] Methylation and Demethylation of both DNA and RNA play vital roles in gene expression (Li et al, Zhang et al, Roundtree et al). DNA demethylation has been shown to affect such plant development as stomatai development, pollen germination, fruit ripening, branching and endosperm as well as embryo development (Li et al., Wei et aL, Wang et al., Duan et al.). DNA methylation and demethylation which can be directed by RNA as occurs with small interfering RNAs direct the cytosine methylation of DNA sequences complementary to the small interfering RNA (Chinnusamy et al). Of particular interest, is work by Yu et al. that showed RNA demethylation increases the yield and biomass of rice and potato. Several patents have been granted for increasing yield by expressing rrf'A demethylase FTO genes from humans, pig, cattle and green algae (U.S. Pat. Nos. 11,046,969; 11,512,322; 11,891,610 and U.S. Pat. App. Pub. No. 2018-0340182).

[0005] There remains a need to identify new variants capable of expressing m6A demethylase FTO genes to provide improved speed of plant development and / or stress tolerance in one or more plant species.Summary of the Invention

[0006] The present invention relates to the demethylation of RNA. Cellular RNA molecules, including tRNAs, rRNAs and mRNAs, have been shown to be involved with 100 types of chemical modifications (Roundtree et al.). Tire most common mRNA modification is N6-methyladenosine (m6A) (Shinde et al.). These modifications have been associated with plant development such as root growth, organ development, fruit ripening and callus induction (Shinde et al., Tang et al., Yu et al.).

[0007] The present invention provides novel engineered protein for conferring enhanced growth. More specifically, the invention relates to recombinant DNA molecules encoding enzymes that confer demethylase activity of RNA.

[0008] More particularly, in a first aspect provided herein are polynucleotides encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid or the full length sequence comprising SEQ ID NOs: 1, 4, or 5.

[0009] In a second aspect, the invention provides recombinant DNA constructs comprising a regulatory element operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence or the full length sequence comprising SEQ ID NOs: 1, 4, or 5. In certain embodiments the regulator}’ element is a heterologous promoter, such as for example, 35S strong constitutive promoter or GOS2 moderately constitutive promoter.

[0010] In another aspect, provided are plant cells, plants, and seeds comprising the polynucleotide encoding a polypeptide or the recombinant DNA construct comprising a regulatory element operably linked to the polynucleotide encoding a polypeptide at least 95% identical to the amino acid of SEQ ID NOs: 1, 4, or 5. In certain embodiments, the regulatory element is a heterologous promoter. In certain embodiments, the plant and / or seed is from a dicot plant. In certain embodiments, the dicot plant is tobacco.

[0011] In yet another aspect, provided are methods for increasing yield in a plant by expressing in a regenerable plant cell a recombinant DNA construct comprising a regulatory element operably linked to a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence of SEQ ID NOs: 1, 4, or 5; and generating the plant, wherein the plant comprises in its genome the recombinant DNA construct. In certain embodiments, the regulator)’ element is a heterologous promoter. In certain embodiments, the plant isa dicot plant. In certain embodiments, the di cot plant is tobacco. In certain embodiments, the increase yield is more biomass.

[0012] In another aspect, the invention provides methods for increasing yield in a plant by introducing in a regenerable plant cell a targeted genetic modification at a genomic locus that encodes a polypeptide comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence or the full length sequence comprising SEQ ID NOs: I, 4, or 5: and generating the plant, wherein the level and / or activity of the encoded polypeptide is increased in the plant. In certain embodiments, the genetic modification is introduced using an expression cassette utilizing 35S promoter driving SEQ ID 2. In certain embodiments, the plant cell is from a dicot plant. In certain embodiments, the dicot plant is tobacco. In certain embodiments, the increased yield is increased biomass.

[0013] In one embodiment, said nucleic acid molecule encoding the mc’A demethylase has at least 90%. preferably at least 95%, more preferably at least 99%, most preferably 100% identity to SEQ ID NOs: 2, 6, or 7.

[0014] The transgenic plant of the present invention exhibits an increased biomass, an increased yield or the combination thereof when compared with a control plant which does not comprise the nucleic acid molecule encoding the mbdemethylase.

[0015] In one embodiment, the plant of the present invention is wheat, rice, com, soybean, canola, tobacco, potato, alfalfa, canola, cotton, sunflower, cannabis, turfgrass or ornamentals. Hie present invention is also directed to a tissue, an organ, a pollen, a seed, a grain, a fruit and a progeny plant of the aforesaid transgenic plant.Brief Description of the Drawings and Sequence Listing

[0016] The patent or patent application file contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0017] FIG. 1 shows the enhanced growth and superior development of three tobacco plants expressing demethylase gene from Ostreococcus RCC809 versus control tobacco plants not expressing the demethylase gene.

[0018] FIG. 2 shows enhanced root development of two independent transgenic tobacco lines expressing demethylase gene from Ostreococcus RCC809 versus control tobacco plants not expressing the demethylase gene.

[0019] FIG. 3 shows faster plant development and earlier flowering of tw o independent transgenic tobacco lines expressing demethylase gene from Ostreococcus RCC809 versus control tobacco plants not expressing the demethylase gene.

[0020] FIG. 4 shows enhanced salt stress tolerance of tobacco seedlings expressing demethylase gene from Ostreococcus RCC809 grown on 200 mM sodium chloride versus control tobacco seedlings not expressing the demethylase gene.

[0021] FIG. 5 shows enhanced salt stress tolerance of tobacco seedlings expressing demethylase gene from Ostreococcus RCC809 grown on 300 mM mannitol versus control tobacco seedlings not expressing the demethylase gene.

[0022] FIG. 6 shows expression cassette.

[0023] FIG. 7 shows enhanced salt stress tolerance of tobacco seedlings expressing demethylase gene from Nitzschia inconspicua, diatom grown on 200 mM sodium chloride versus control tobacco seedlings not expressing the demethylase gene.

[0024] FIG. 8 shows enhanced salt stress tolerance of tobacco seedlings expressing demethylase gene from Aphanomyces cochlioides, spinach pathogen grown on 200 mM sodium chloride versus control tobacco seedlings not expressing the demethylase gene.

[0025] FIG. 9 shows enhanced leaf size of an embodiment of the invention compared to wild type and vector control.

[0026] FIG. 10A shows enhanced root size of an embodiment of the invention compared to wild type and vector control.

[0027] FIG. 10B shows enhanced root size of an embodiment of the invention compared to wild type and vector control.

[0028] SEQ ID NO: 1 Ostreococcus RCC809 demethylase amino acid sequence.

[0029] SEQ ID NO: 2 Ostreococcus RCC809 demethylase nucleotide sequence.

[0030] SEQ ID NO: 3 Ostreococcus RCC809 FTO gene in pF ATI.

[0031] SEQ ID NO: 4 Nitzschia inconspicua. diatom demethylase amino acid sequence.

[0032] SEQ ID NO: 5 Aphanomyces cochlioides, spinach pathogen demethylase amino acid sequence.

[0033] SEQ ID NO: 6 Nitzschia inconspicua, diatom demethylase nucleic acid sequence.

[0034] SEQ ID NO: 7 Aphanomyces cochlioides, spinach pathogen demethylase nucleic acid sequence.

[0035] SEQ ID NO: 8 Nitzschia inconspicua, diatom demethylase complete vector and T- DNA sequence.

[0036] SEQ ID NO: Aphanomyces cochlioides. spinach pathogen demethylase complete vector and T-DNA sequence.

[0037] SEQ ID NO: 10 Nitzschia inconspicua, diatom demethylase expression cassette SEQ ID NOS:3 and 5.

[0038] SEQ ID NO: 11 Aphanomyces cochlioides, spinach pathogen demethylase expression cassette SEQ ID NOS: 4 and 6.

[0039] The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.

[0040] Tire sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.825. The sequence descriptions comprise the three letter codes for amino acids as defined in 37 C.F.R. §§ 1.821 and 1.825, which arc incorporated herein by reference.Detailed DescriptionI. CompositionA. Polynucleotides and Polypeptides

[0041] The present disclosure provides polynucleotides encoding polypeptides. Accordingly, as used herein “polypeptide,” “protein,” or the like, refers to a protein represented by a SEQ ID NOs.1, 4. and 5. One aspect of the disclosure provides a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 90-99 %, and preferably 95-99 % identical to the amino acid sequence of SEQ ID NOs: 1 , 4, or 5, or 100% identical to each of those sequences.

[0042] m°A demethylase and the nucleic acids encoding said demethylase may be used to produce the transgenic plant of the present invention. The m°A demethylase used by the present invention was identified in Ostreococcus sp. RCC809; Nitzschia inconspicua, diatom,' and Aphcmomyces cochlioides, spinach pathogen, which differ from the m6A demethylase disclosed in U.S. Pat. Nos. 11,046.969; 11,512,322; 11,891,610 and U.S. Pat. App. Pub. No. 2018-0340182. Hie irf'A demethylase of SEQ ID NO: 1 has only 30%, 29%, 30% and 70% protein homology to the human (’969 patent, SEQ ID NO: 1), pig (’969 patent, SEQ ID NO: 2), cattle (‘969 patent, SEQ ID NO: 3) and green alga (‘969 patent, SEQ ID NO: 4). The rn6A demethylase of SEQ ID NO: 4 has only 28%, 29%, 28% and 32% identity to the human (‘969 patent, SEQ ID NO: 1), pig (‘969 patent, SEQ ID NO: 2), cattle (‘969 patent, SEQ ID NO: 3) and green alga (‘969 patent, SEQ ID NO: 4) and SEQ ID NO:5 has 29%, 29%, 28% and 32% identity to the human (‘969 patent, SEQ ID NO: 1), pig (‘969 patent, SEQ ID NO: 2), cattle (‘969 patent, SEQ ID NO: 3) and green alga (‘969 patent. SEQ ID NO: 4). The present inventors have observed qualitative improvements in 1) speed of plant development and 2) stress tolerance (osmotic) for the m°A demethylase of the present invention.

[0043] As used herein “homolog” means a protein that perform the same biological function. Homologs arc expressed by homologous genes. Homologous genes include naturally occurring alleles and artificially created variants. Homologs are proteins that when aligned have at least 90%, preferable at least 95% identity over the full length of a protein identified as increasing the yield and / or biomass or stress tolerance of plants when expressed in plant cells.

[0044] Homologs are identified by comparison of amino acid sequence, e g. manually or by use of a computer-based tool using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A further aspect of the homologs encoded by DNA useful in the transgenic plants of tire invention are those proteins that differ from a disclosed protein as the result of deletion or insertion of one or more amino acids in a native sequence.

[0045] As used herein, ‘‘percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, for example nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by¬ sequences of the two aligned segments divided by the total number of sequence components in the reference segment over a window of alignment which is the smaller of the full test sequence or the full reference sequence. “Percent identity” (“% identity”) is the identity fraction times 100.

[0046] The m6A demethylase used in the present invention may be SEQ ID NOs: 1, 4, or 5, or the homolog thereof. Said homolog has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1, 4, or 5.

[0047] The nucleic acid encoding the m°A demethylase used in the present invention may be any one of SEQ ID NOs: 2, 6, or 7, or the homologous gene thereof. Said homologous gene has at least 90%. 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2, 6, or 7.

[0048] Tire nucleic acid encoding the mbA demethylase defined herein may not be a full-length nucleic acid. A part of the nucleic acid encoding the m6A demethylase defined herein may be prepared by making one or more deletions from the full-length nucleic acid.

[0049] All or a portion of the nucleic acids of the present invention may be synthesized using codons preferred by a selected host. Species-preferred codons may be determined, for example, from tire codons used most frequently in tire proteins expressed in a particular host species. Therefore, the nucleic acids of the present invention include those obtained by making codon-optimization to natural m6A demethylase for the expression in plants. Other modifications of the nucleotide sequences may result in mutants having slightly altered activity.

[0050] The present invention is directed to a transgenic plant in which a gene encoding the aforesaid m£A demethylase or a homolog thereof is introduced, or a progeny plant thereof. Tire present invention is also directed to a cell, a tissue, an organ, a pollen, a seed, a grain, a tuber, a stolon, a rhizome or a fruit of said plant.

[0051] ■‘Introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment may be incorporated into the genome of tire cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0052] “Plant” includes reference to whole plants, plant organs, plant tissues, seeds and plant cells and progeny of same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, stolons, rhizomes, tubers, fruits, gametophytes, sporophytes, pollen, and microspores. “Progeny” comprises any subsequent generation of a plant.

[0053] According to the use, the plants of the present invention may be food crops, economic crops, vegetable crops, fruits, flowers, grasses, trees, industrial raw material crops, feed crops or medicine crops. Specifically, said food crops include rice, maize, soybean, beans, yams, potato, barley, broad bean, wheat, barley, millet, rye, oat, sorghum, etc.; Said economic crops include oil tea, rape, rapeseed, flax, false flax (Camelina sativa), peanut, oil flax (Linum usitatissimum), marijuana (Cannabis sativa), sunflower, tobacco, cotton, beet, sugarcane, etc.; said vegetable crops include radish, Chinese cabbage, tomato, cucumber, hot pepper, carrot, etc.; said fruits include pear, apple, walnut, cherry, strawberry, blueberry, raspberry, blackberry, jujube or peach; said flowers include flowers for view, for example, orchid, Chrysanthemum, carnation, rose, petunia, geranium, vinca, impatiens, green plants, etc., grasses for example creeping bentgrass, tall fescue, ryegrass, St. Augustinegrass, zoysia, bermudagrass and trees include Populus, Hevea brasiliensis, Taxus chinensis, and those for urban greening or those living in deserts and harsh conditions such as drought; said industrial raw material crops include Russian dandelion, guayule, Jatropha curcas, etc., said feed crops include the foodstuff for livestock, such as alfalfa etc.; said drug crops include Ginseng, Angelica and Ganoderma, etc.

[0054] “Transgenic plant” includes reference to a plant which comprises within its genome a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. Tire heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct.

[0055] “Heterologous” with respect to sequence means a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0056] The DNA construct of the present invention comprises a nucleic acid molecule encoding tire m°A demethylase protein described herein. The construct may comprise the nucleic acid molecule encoding the m6A demethylase protein described herein optionally operably linked to a promoter sequence which functions in a host cell. Other construct components may include additional regulator} elements, such as 5' leaders and introns for enhancing transcription, 3' untranslated regions (such as polyadenylation signals and sites), DNA for transit, signal peptides, or one or more selective maker genes.

[0057] As used herein, “promoter” means regulatory DNA for initializing transcription. A plant promoter is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. is it well known that Agrobacterium promoters are functional in plant cells. Thus,plant promoters include promoter DNA obtained from plants, plant viruses and bacteria such as Agrobacterium and Bradyrhizobium bacteria. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters that initiate transcription only in certain tissues are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, or certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most conditions. Promoters useful in the present invention are not specifically limited. Those skilled in the art may select suitable promoters according to their knowledge.

[0058] As used herein “operably linked” means the association of two or more DNA fragments in a DNA construct so that the function of one, e.g. protein-encoding DNA, is controlled by the other, e.g. a promoter.

[0059] The DNA construct generally includes a selective maker gene. Selective marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to pennit cell survival. Cells may be tested further to confirm stable integration of tire exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin (nptll), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (aroA or EPSPS). Examples of such selective markers are illustrated in U.S. Pat. Nos.5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Selective markers which provide an ability to visually identify transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.

[0060] The introduction of the recombinant DNA construct into plants may be carried out by any suitable technique, including but not limited to direct DNA uptake, chemical treatment, electroporation, microinjection, cell fusion, infection, vector mediated DNA transfer, bombardment, or Agrobacterium mediated transformation. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs will include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.

[0061] In general, it is useful to introduce recombinant DNA randomly, i.e. at a non-specific location, in the genome of a target plant line. In special cases it may be useftd to target recombinant DNA insertion to achieve sitc-spccific integration, for example to replace an existing gene in the genome, to use an existing promoter in tire plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site-specific recombination systems exist which are known to function in plants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.

[0062] Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. ‘‘Media" refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and tire like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, for example various media and recipient target cells, transformation of immature embryo cells and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526, which are incorporated herein by reference.

[0063] The development or regeneration of plants including the foreign, exogenous isolated nucleic acid fragment that encodes a protein of interest is well known in tire art. The regenerated plants are self-pollinated to provide homozygous transgenic plants. Otherwise, pollen obtained from the regenerated plants is crossed to seed-grown plants of agronomically important lines. Conversely, pollen from plants of these important lines is used to pollinate regenerated plants. A transgenic plantof the present invention including the nucleic acid molecule encoding the m6A demethylase is cultivated using methods well known to one skilled in the art.

[0064] The seeds of transgenic plants can be harvested from fertile transgenic plants and be used to grow progeny generations of transformed plants of this invention including hybrid plants line for selection of plants having an enhanced trait. In addition to direct transformation of a plant with the nucleic acid molecule encoding the m' A demethylase. transgenic plants can be prepared by crossing a first plant having the nucleic acid molecule encoding the nTA demethylase with a second plant lacking the nucleic acid molecule. For example, the nucleic acid molecule encoding the m’ A demethylase can be introduced into the first plant line that is amenable to transformation to produce a transgenic plant which can be crossed with a second plant line to introgress the nucleic acid molecule encoding tire m6A demethylase into the second plant line. Tire transgenic plant derived from the plant cell of the present invention is cultivated to produce increased yield and / or biomass compared with a control plant. As used herein a “control plant” means a plant that does not contain the nucleic acid molecule encoding the m6A demethylase. A control plant is to identify and select a transgenic plant that has increased yield and / or biomass. A suitable control plant can be a non-transgenic plant of the parental line used to generate a transgenic plant, i.c. devoid of the nucleic acid molecule encoding the m6A demethylase. A suitable control plant may in some cases be a progeny of ahemizygous transgenic plant line that does not contain the nucleic acid molecule encoding the nf’A demethylase, known as a negative segregant.

[0065] The “yield” of the transgenic plant described herein means the harvest amount of the product desired by the cultivation. The standards for evaluating the yields of different plants are different. For example, the subject of the evaluation of the yields of cereal crops (rice, wheat, maize, etc.), beans and oil crops (soybean, peanut, rape, etc.) is the seed (grain); that of cotton is the seed cotton or tire lint cotton: that of yam crops (sweet potato, potato, cassava, etc.) is the tuberous root or tuber; that of bast fiber crops is the fiber of stems or the fiber of leaves; that of sugarcane is the stem; that of beet is the root; that of tobacco is the leaf; that of green manure crops (alfalfa, trefoil, etc.) is the stem and leaf, etc. The meaning of the yield of the same plant differs when it is cultivated for different purposes. For example, when maize is cultivated as food and fine feed crop, the yield is the harvest amount of grains, and when it is cultivated as silage, the yield includes the total harvest amount of stems, leaves and ears. The increased yield of the transgenic plant of the present invention may be measured by many means, including measuring weight, seed number per plant, seed weight, tuber weight, seed number per unit area (i .e. seeds, or weight of seeds, per acre), bushels per acre, tons per acre, kilo per hectare.

[0066] Those skilled in the art can determine the meaning of the yield for each plant and the standard for evaluating it according to the knowledge in the art.

[0067] Biomass means the total mass of the existing organic materials of an organism. It is expressed as dry weight, fresh weight, tiller number, etc. in the present invention. The increased biomass of the transgenic plant of the present invention may be measured by many means, including measuring weight, dry weight or fresh weight of the overground parts per plant, tiller number, dry weight of the overground parts per unit area (i.e. dry weight or fresh weight, per acre), bushels per acre, tons per acre, kilo per hectare.EXAMPLES

[0068] The present invention is further illustrated in the following Examples. It should be understood that these Examples, while indicating embodiments of the invention, are given by way of illustration only. From tire above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

[0069] Furthermore, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0070] EXAMPLE 1

[0071] Transformation Method of Tobacco

[0072] 1. Materials:

[0073] Tobacco variety Burley 21: Agrobacterium'. LBA4404: Ostreococcus RCC809 demethylase gene in pMGl driven by a 35 S promoter.

[0074] 2. Tobacco Transformation

[0075] Agrobacterium cultures were grown from a single colony for 2-3 days at 28oC in YEP (Y east Extract Peptone Dextrose) medium containing 50 mg / L kanamycin, 25 mg / L rifamycin and 25 mg / L streptomycin. Explant material was obtained by growing sterilized seeds in magenta boxes for 3-5 weeks. Co-cultivation of sterile tobacco leaf material cut in 2 mm segments with the agrobacterium culture that has been diluted to 10-6 colony forming units for three days. Wash explants in MS + 1 mg / L BA + 0.1 mg / L NAA + 250 mg / L carbenicillin liquid medium for 5 to 15min Selection was performed on solid plants containing 50 mg / L kanamycin, 25 mg / L rifamycin and 25 mg / L streptomycin in YEP medium. Transgenic plants were regenerated in MS + 1 mg / L IBA + 150 mg / L Kn solid medium.

[0076] 3. Seedling Vigor Screening

[0077] Tobacco seeds were sown on growing media containing peat moss and perlite. The seeds and seedlings were watered as needed with 125 ppm Miracle-Gro fertilizer and maintained at 21oC under 18 hours of LED grow lights.

[0078] 4. Enhanced Root Development

[0079] Four-week-old tobacco seedlings were grown in in 2.7 liter pots. The plants were watered as needed with 125 ppm Miracle-Gro fertilizer and maintained at 21oC under 18 hours of LED grow lights.

[0080] 5. Enhanced Plant Development and Faster Flowering

[0081] Four-week-old tobacco seedlings were grown in 2.7-liter pots. Tire plants were watered as needed with 125 ppm Miracle-Gro fertilizer and maintained at 21oC under 18 hours of LED grow lights.

[0082] 6. Enhanced Salt Tolerance

[0083] Tobacco seedlings were sterilized and grown on MS medium + 200 mM NaCl.

[0084] 7. Enhanced Osmotic Tolerance

[0085] Tobacco seedlings were sterilized and grown on MS medium + 300 mM mannitol.Complete vector and T-DNA sequences

[0086] SEQ ID NO: 3 - Ostreococcus RCC809 FTO gene in pFATl

[0087] 3874 bp linear DNA between T DNA right and left borders

[0088] REFERENCE 1 (bases 1 to 3874)

[0089] FEATURES Location / Qualifiers

[0090] mise feature 1..26

[0091] gene="Left Border T-DNA repeat"

[0092] misc_feature 27..92

[0093] gene-' Cloning site"

[0094] misc feature complement(93..336)

[0095] gene="CaMV 3' UTR"

[0096] gene complement(337..1131)

[0097] gene="nptll"

[0098] product="Kan resistance"

[0099] misc feature complement(l 132..1942)

[0100] gene="2 x CaMV 35S promoter"

[0101] product="Strong promoter"

[0102] misc feature 1943..2007

[0103] gene="Cloning site"

[0104] product-'Multiple cloning site"

[0105] misc feature 2008..2352

[0106] gene="CaMV 3 S promoter"

[0107] misc feature 2353..2358

[0108] gene=" Cloning site"

[0109] CDS 2359..3609

[0110] gene="Ostreococcus RCC809 FTO"

[0111] product- 'RCC809 FTO gene"

[0112] misc feature 3610..3848

[0113] gene="CaMV 3' UTR"

[0114] misc feature 3849..3874

[0115] gene="Right Border T-DNA repeat"

[0116] EXAMPLE 2

[0117] Transformation Method of Tobacco

[0118] 1. Materials:

[0119] Tobacco variety Burley 21; Agrobacteriunr. LBA4404; Nitzschia inconspicua, diatom and Aphanomyces cochlioides, spinach pathogen demethylase gene in pMGl driven by a 35 S promoter.

[0120] 2. Tobacco Transfonnation

[0121] Agrobacterium cultures were grown from a single colony for 2-3 days at 28oC in YEP (Yeast Extract Peptone Dextrose) medium containing 50 mg / L kanamycin, 25 mg / L rifamycin and 25 mg / L streptomycin. Explant material was obtained by growing sterilized seeds in magenta boxes for 3-5 weeks. Co-cultivation of sterile tobacco leaf material cut in 2 mm segments with the agrobacterium culture that has been diluted to 10-6 colony forming units for three days. Wash explants in MS + 1 mg / L BA + 0.1 mg / L NAA + 250 mg / L carbenicillin liquid medium for 5 to 15 min Selection was performed on solid plants containing 50 mg / L kanamycin, 25 mg / L rifamycin and 25 mg / L streptomycin in YEP medium. Transgenic plants were regenerated in MS + 1 mg / L IBA + 150 mg / L Kn solid medium.

[0122] 3. Enhanced Salt Tolerance

[0123] Tobacco seedlings were sterilized and grown on MS medium + 200 rnM NaCl.

[0124] 4. Seedling Vigor Screening

[0125] Tobacco seeds arc sown on growing media containing peat moss and perlite. The seeds and seedlings are watered as needed with 125 ppm Miracle-Gro® fertilizer and maintained at 21oC under 18 hours of LED grow lights.

[0126] 5. Enhanced Root Development

[0127] Pour-week-old tobacco seedlings are grown in in 2.7 liter pots. The plants are watered as needed with 125 ppm Miracle-Gro ® fertilizer and maintained at 21oC under 18 hours of LED grow lights.

[0128] 6. Enhanced Plant Development and Faster Flowering

[0129] Four-week-old tobacco seedlings are grown in 2.7-liter pots. The plants are watered as needed with 125 ppm Miracle-Gro® fertilizer and maintained at 21oC under 18 hours of LED grow lights.

[0130] EXAMPLE 3

[0131] SEQ ID NO: 8 Nitzschia inconspicua, diatom demethylase complete vector and T- DNA sequence.

[0132] LOCUS Nitzachia inconspicua FTO 4279 bp

[0133] DNA linear SYN 20-AUG-2025

[0134] ACCESSION M7_insert

[0135] REFERENCE 1 (bases 1 to 4279)

[0136] AUTHORS Self

[0137] JOURNAL Unpublished.

[0138] FEATURES Location / Qualifiers

[0139] misc feature 1..26

[0140] gene="Left Border T-DNA repeat"

[0141] misc_feature 27..92

[0142] gene- 'Cloning site"

[0143] misc feature complement(93..336)

[0144] gene="CaMV 3' UTR"

[0145] gene complement^ 37..1131 )

[0146] gene="nptl 1"

[0147] product="Kan resistance"

[0148] misc feature complement 1132..1942)

[0149] gene="2 x CaMV 35S promoter"

[0150] product- ' Strong promoter"

[0151] misc feature 1943..2007

[0152] gene="Cloning site"

[0153] product- 'Multiple cloning site"

[0154] misc feature 2008..2352

[0155] gene="CaMV 35S promoter"

[0156] misc_feature 2353..2358

[0157] gene="Cloning site'"

[0158] CDS 2359..4011

[0159] gene="Nitxschia inconspicua demathylase"

[0160] product- ' FTO demethylase"

[0161] misc feature 4012..4019

[0162] gene="Cloning site"

[0163] misc feature 4020..4253

[0164] gene="'CaMV 3' UTR"

[0165] misc feature 4254..4279

[0166] gene="Right Border T-DNA repeat"

[0167] EXAMPLE 4

[0168] Complete vector and T-DNA sequences

[0169] SEQ ID NO: 9 Aphanomyces cochlioides, spinach pathogen demethylase complete vector and T-DNA sequence.

[0170] LOCUS Aphanomyces cochlioides FTO 4162 bp DNA linear

[0171] SYN 20-AUG-2025

[0172] ORGANISM Aphanomyces cochlioides

[0173] FEATURES Location / Qualifiers

[0174] misc feature 1..26

[0175] / gene="Left Border T-DNA repeat"

[0176] misc_feature 27..92

[0177] / gene="Cloning site"

[0178] mi sc feature complement(93..336)

[0179] / gene="CaMV 3' UTR"

[0180] gene complement(337..1131)

[0181] / gene- 'nptll"

[0182] / product="Kan resistance"

[0183] misc feature complement^ 132..1942)

[0184] / gene="2 x CaMV 35S promoter"

[0185] / product-' Strong promoter"

[0186] misc feature 1943..2007

[0187] / gene="Cloning site"

[0188] / product- 'Multiple cloning site"

[0189] misc feature 2008..2352

[0190] / gene="CaMV 35S promoter"

[0191] misc_feature 2353

[0192] / gene="Cloning site'"

[0193] CDS 2359..3897

[0194] / gene="Aphanomyces cochlioides FTO"

[0195] / product- 'Aphanomyces cochlioides FTO"

[0196] misc feature 3903..4136

[0197] / gene="'CaMV 3' UTR"

[0198] misc feature 4137..4162

[0199] / gene="Right Border T-DNA repeat'

[0200] EXAMPLE 5

[0201] Tobacco variety Burley 21 was transformed with Aphanomyces cochlioides, spinach pathogen demethylase gene in pMGl driven by a 35S promoter as described herein. Fig. 9 shows how this transformation dramatically increased leaf size relative to tire wild type and vector control plants. Figs. 10A and 10B show the visual improvement in root size as the increase in measured root weight relative to wild type and vector control plants.

[0202] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all U.S. and foreign patents and patent applications, are specifically and entirely hereby incorporated herein by reference. It is intended that the specification and examples be considered exemplary only, with the true scope and spirit of the invention indicated by the following claims.

Claims

What is claimed is:

1. A transgenic plant, seed, cell or plant part comprising a polynucleotide capable of expressing a recombinant polypeptide that is at least 95% identical to an amino acid sequence of SEQ ID NOs: 1, 4, or 5, the polynucleotide being operably linked to a plant expressible promoter, wherein the transgenic plant, seed, cell or plant part has enhanced salt stress tolerance compared to a control plant, seed, cell, or plant part that does not express the recombinant polypeptide.

2. The transgenic plant, seed, cell or plant part of claim 1 wherein the recombinant polypeptide comprises an amino acid of SEQ ID NOs: 1, 4, or 5.

3. A method of increasing the yield of a plant comprising transforming said plant with a polynucleotide capable of expressing a recombinant polypeptide that is at least 95% identical to an amino acid sequence of SEQ ID NOs: 1, 4, or 5, the polynucleotide being operably linked to a plant expressible promoter, wherein the plant has enhanced salt stress tolerance compared to a control plant that does not express the recombinant polypeptide.

4. The method of Claim 3 further comprising expressing the protein encoded by the polynucleotide.

5. The transgenic plant, seed, cell or plant part of claim 1 that is, or is from, a dicot plant.

6. Tire transgenic plant, seed, cell or plant part of claim 5, wherein the dicot plant is selected from soybean, canola, tobacco, potato, alfalfa, cotton, sunflower, or cannabis.

7. Tire transgenic plant, seed, cell or plant part of claim 5, wherein the dicot plant is tobacco.

8. The transgenic plant, seed, cell or plant part of claim 1 that is, or is from, a monocot plant.

9. The transgenic plant, seed, cell or plant part of claim 8, wherein the monocot plant is selected from wheat, rice, com, turfgrass or ornamental grass.

10. A transgenic plant, seed, cell or plant part comprising a polynucleotide that is at least 95% identical to a nucleic acid sequence of SEQ ID NOs:3, 6, or 7, the polynucleotide being operably linked to a plant expressible promoter, wherein the transgenic plant, seed, cell or plant part has enhanced salt stress tolerance compared to a control plant, seed, cell, or plant part that does not express the recombinant polypeptide.

11. The transgenic plant, seed, cell or plant part of claim 10 comprising a polynucleotide that comprises a nucleic acid of SEQ ID NOs:3, 6. or 7.

12. A method of increasing the yield of a plant comprising transforming said plant with a polynucleotide that is at least 95% identical to a polynucleotide sequence of SEQ ID N0s:3, 6, or 7, the polynucleotide being operably linked to a plant expressible promoter, wherein the plant has enhanced salt stress tolerance compared to a control plant that does not express the recombinant polypeptide.

13. The method of Claim 12 further comprising expressing the protein encoded by the polynucleotide.

14. The transgenic plant, seed, cell or plant part of claim 10 that is, or is from, a dicot plant.

15. The transgenic plant, seed, cell or plant part of claim 14, wherein the dicot plant is selected from soybean, canola, tobacco, potato, alfalfa, cotton, sunflower, or cannabis.

16. Tire transgenic plant, seed, cell or plant part of claim 14, wherein the dicot plant is tobacco.

17. Tire transgenic plant, seed, cell or plant part of claim 10 that is, or is from, a monocot plant.

18. Tire transgenic plant, seed, cell or plant part of claim 17, wherein the monocot plant is selected from wheat, rice, com, turfgrass or ornamental grass.

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