Inducible flowering for fast generation times in maize and sorghum

Abstract

A method of producing faster flowering times in corn and sorghum plants is presented herein. Corn and sorghum plants comprising a non-native flowering gene that flower faster by at least three developmental leaves than control plants are also presented herein.

Claims

1. A method of producing corn or sorghum plants with earlier flowering times comprising: a. transferring a FT gene into a plant or plant cell to produce a maize or sorghum plant comprising a non-native FT gene or protein; b. expressing said FT gene or protein of step (a) in one or more embryos, seeds or seedlings to induce early flowering, wherein said early flowering occurs at least 3 developmental leaves earlier than isogenic control plants lacking said FT gene or protein.

2. The method of claim 1, wherein expression of the FT gene or protein in step (b) is inducible.

3. The method of claim 2, wherein expression of the FT gene in step (b) is chemically inducible with a ligand that binds the ligand binding-activation domain of an ecdysone receptor, wherein the ligand is selected from the group consisting of methoxyfenozide, tebufenozide, and other compounds.

4. The method of claim 2, wherein expression of the FT gene in step (b) is chemically inducible with a ligand that binds the ligand binding-activation domain of chimeric transcription factor, wherein the ligand is selected from the group consisting tetracycline, estradiol, dexamethasone, alcohol, copper, zinc, or cadmium.

5. The method of claim 1, wherein expression of the FT gene or protein in step (b) is constitutively expressed from a weak constitutive promoter.

6. The method of claim 1, wherein expression of the FT gene or protein in step (b) is expressed from a phloem active promoter expressed at higher levels in a plant than in embryogenic callus.

7. The method of claim 1, wherein the FT gene in step (b) encodes a FT protein with at least 85%, 90%, or 95% homology to the sequence of SEQ ID No. 2.

8. The method of claim 1, wherein the FT gene in step (b) encodes a FT protein that is more homologous to SEQ ID No. 2 than to ZCN8 in the in the C-terminal region comprising 50% of the proteins.

9. The method of claim 1, wherein the FT gene in step (b) encodes a FT protein permutein.

10. The method of claim 1, wherein expression of an endogenous FT gene is altered by gene editing to form a non-native FT gene sequence comprising an altered promoter that causes early flowering relative to control non-altered parental plants.

11. A corn or sorghum plant or progeny thereof produced by the method of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1. Line drawing of genetic elements of plasmid pUbiqFT. Genetic elements: CaMV 35S Pro: Cauliflower Mosaic Virus 35S Promoter; Hsp70 int: intron of maize Hsp70 gene; Gfp-NptII: green fluorescent-neomycin phosphotransferase II fusion protein; T35S: terminator or 3 polyadenylation region of Cauliflower Mosaic Virus; ZmUbiq1/int: Zea Mays Ubiquitin 1 (Ubi-1) promoter; FT: synthetic Flowering Time (SEQ ID No. 3); Nos3: Nopaline Synthase: terminator or 3 polyadenylation region; RBr: Agrobacterium Ti plasmid right border; pPZP: binary vector pPZP backbone; rrnBTIT2: E. coli ribosomal terminator from rrnB gene.

[0026] FIG. 2. Line drawing of genetic elements of plasmid piFT1. Genetic elements as in FIG. 1 description and the following elements: FMV: Figwort Mosaic Virus 34S promoter; XEV: modified inducible chimeric transcription factor from XEV system; PinII3: terminator or 3 polyadenylation region of potato PinII gene; OpPro: LexA operator with minimal CaMV 35S promoter; Adh1int: first intron of maize Adh1 gene;

[0027] FIG. 3. Floral structures present in FT containing corn plants. The floral structures from two independently transformed plants are shown. These were removed from corn plants with three to five leaves (not shown) that were regenerating from embryogenic tissues in 20 cm high petri dishes. The ovules and silks are designated and were nearly surrounded by wide leaf ear husk type structures that were dissected away for access to the ovules.

DEFINITIONS

[0028] As used herein, the phrase flowering refers to formation of either male anthers or female ovules and stigma or complete (male and female) flowers formed on a plant.

[0029] As used herein, the phrase developmental leaves refers to the number of leaves formed prior to the time of flowering on a plant. Flowering can be the ear or tassel or head in the case of sorghum.

[0030] As used herein, the phrase 3 developmental leaves refers to difference of three leaves in the number of leaves formed prior to the time of flowering on a first plant relative to a second plant.

[0031] As used herein, the phrase gene refers to a DNA genetic element that when in a cell causes transcription of the DNA into RNA. A gene typically comprises a promoter, transcribed region, and an associated RNA termination and/or polyadenylation processing region. Some genes may lack a RNA termination and/or polyadenylation region and still produce RNA.

[0032] As used herein, the phrases commercially synthesized or commercially available DNA refer to the availability of any sequence of 15 bp up to 2000 bp in length or longer from DNA synthesis companies that provide a DNA sample containing the sequence submitted to them.

[0033] As used herein, the term F1 refers to the first progeny of two genetically or epigenetically different plants. F2 refers to progeny from the self pollination of the F1 plant. F3 refers to progeny from the self pollination of the F2 plant. F4 refers to progeny from the self pollination of the F3 plant. F5 refers to progeny from the self pollination of the F4 plant. Fn refers to progeny from the self pollination of the F(n1) plant, where n is the number of generations starting from the initial F1 cross. Crossing to an isogenic line (backcrossing) or unrelated line (outcrossing) at any generation will also use the Fn notation, where n is the number of generations starting from the initial F1 cross.

[0034] Homology as used herein refers to sequence identity or similarity between a reference sequence and at least a fragment of a second sequence. Homology may be identified by any method known in the art, preferably, by using the BLAST or BLASTP or CLUSTAL Omega tool to compare a reference sequence or sequences to a single second sequence or fragment of a sequence or to a database of sequences. Homology includes alignment with a permutein of a sequence or protein such that alignment occurs in at least two blocks due to the circularization/opening at different N and C termini that occurs in a permutation of a gene or protein in a permutein. Optionally, homology has 70%, 75%, 80/%, 85%, 90%, 95%, 990/% or 100% identity or similarity over a specified region, or, when not specified, over the entire sequence including the case of two regions for comparison to a permutein. The specified or entire sequence length is at least 50 amino acids or longer. As described below, BLAST (or BLASTP) or CLUSTAL Omega will compare sequences based upon percent identity and similarity.

[0035] As used herein similarity or similar refers to non-identical amino acids within the same group, where the groups are: aliphatic (Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or Sulfur/Selenium-containing (Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic (Proline); Aromatic (Phenylalanine, Tyrosine, Tryptophan); Basic (Histidine, Lysine, Arginine); or Acidic and their Amides (Aspartate, Glutamate, Asparagine, Glutamine).

[0036] The terms identical in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are substantially identical if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 29% identity, optionally 30/%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol 215(3)-403-410, respectively. The BLASTN program (for nucleotide sequences) or BLASTP program (for amino acid sequences) or CLUSTAL Omega are suitable for most alignments.

[0037] As used herein, the phrase loss of function refers to a diminished, partial, or complete loss of function.

[0038] The phrase operably linked as used herein refers to the joining of nucleic acid sequences such that one sequence can provide a required function to a linked sequence. In the context of a promoter, operably linked means that the promoter is connected to a sequence of interest such that the transcription of that sequence of interest is controlled and regulated by that promoter. When the sequence of interest encodes a protein and when expression of that protein is desired, operably linked means that the promoter is linked to the sequence in such a way that the resulting transcript will be efficiently translated. If the linkage of the promoter to the coding sequence is a transcriptional fusion and expression of the encoded protein is desired, the linkage is made so that the first translational initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made so that the first translational initiation codon contained in the 5 untranslated sequence associated with the promoter is linked such that the resulting translation product is in frame with the translational open reading frame that encodes the protein desired. Nucleic acid sequences that can be operably linked include, but are not limited to, sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5 untranslated regions, introns, protein coding regions, 3 untranslated regions, polyadenylation sites, and/or transcriptional terminators), sequences that provide DNA transfer and/or integration functions (i.e., site specific recombinase recognition sites, integrase recognition sites), sequences that provide for selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker functions (i.e., reporter genes), sequences that facilitate in vitro or in vivo manipulations of the sequences (i.e., polylinker sequences, site specific recombination sequences, homologous recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomous replication sequences, centromeric sequences).

[0039] As used herein, the term progeny refers to any one of a first, second, third, or subsequent generation obtained from a parent plant if self-pollinated or from parent plants if obtained from a cross, or through any combination of selfing and crossing. Any materials of the plant, including but not limited to seeds, tissues, pollen, and cells can be used as sources of RNA or DNA for determining the status of the RNA or DNA composition of said progeny.

[0040] As used herein, the phrase reference plant refers to a parental plant or progenitor of a parental plant prior to epigenetic modification, but otherwise genetically the same as the candidate or test plant to which it is being compared.

[0041] As used herein, the terms self, selfing, or selfed refer to the process of self pollinating a plant.

[0042] As used herein, the term transgene or transgenic refers to any recombinant DNA that has been transiently introduced into a cell or stably integrated into a chromosome or minichromosome that is stably or semi-stably maintained in a host cell. In this context, sources for the recombinant DNA in the transgene include, but are not limited to, DNAs from an organism distinct from the host cell organism, species distinct from the host cell species, varieties of the same species that are either distinct varieties or identical varieties, DNA that has been subjected to any in vitro modification, in vitro synthesis, recombinant DNA, and any combination thereof. The terms transgene or transgenic include inserting or changing DNA sequences at endogenous genes to alter their expression or function through any non-natural process.

[0043] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

EXAMPLES

Example 1. Design and Construction of a Synthetic FT Gene for Maize and Sorghum

[0044] The protein sequence of the corn ZCN15 FT homolog was the starting point for the design of a new FT protein designed to function in corn or sorghum. The maize ZCN15 and a rice FT (SEQ ID No. 1) protein sequences were aligned by BLASTP and non-conservative amino acid changes present in ZCN15 were changed to the rice amino acid at that position. The resulting synthetic corn FT protein sequence is SEQ ID No. 2.

Example 2. Design and Construction of a Synthetic DNA Encoding Synthetic FT Gene for Maize

[0045] A synthetic nucleic acid encoding the corn synthetic FT protein of Example 1 was designed by reverse translating a protein sequence to the possible codons encoding each amino acid at each position and then picking codons that are enriched in maize genes. The resulting synthetic DNA encoding synthetic corn is SEQ ID No. 3. Other alternative codons could be used to in the design of nucleic acid coding regions to encode the same protein.

Example 3. Moderate to Strong Expression of Synthetic FT Prevents Plant Regeneration in Embryogenic Corn Cells

[0046] A plasmid construct pUbiqFT using the maize ubiquitin promoter with its intron to express the synthetic FT coding region (SEQ ID No. 3) and a selectable marker for corn transformation were made (FIG. 1). This plasmid was transformed into immature B104 corn embryos via Agrobacterium mediated transformation and G418 selection for GFP-NptII calli, and transgenic embryogenic callus were obtained. These embryogenic calli were unable to produce transgenic corn plants in the regeneration protocol. We concluded constitutive moderate to high levels of FT interfere with plant regeneration.

Example 4. Low Constitutive and Inducible Expression of Synthetic Corn FT in Corn

[0047] An inducible FT expression vector was made using a modification of the XVE estradiol inducible gene expression system (see U.S. Pat. No. 6,784,340 and Zuo et al., The Plant Journal (2000) 24(2), 265-273). A plasmid map of piFT1 using the modified XVE system used here to express the FT coding region of Example 2 is shown in FIG. 2 and the sequence of the genes in piFT1 are in SEQ ID No. 4. This piFT1 plasmid was transformed into immature B104 corn embryos via Agrobacterium mediated transformation and G418 selection for GFP-NptII calli, and transgenic embryogenic callus obtained. These embryogenic calli were able to regenerate to transgenic corn shoots with roots in the regeneration protocol.

[0048] Two independently transformed regenerating transgenic plants formed flower reproductive structures while still in petri dishes while the shoots were in the three to five leaf stage. Ovules on regenerating shoots in culture have not been observed before in any maize transformations in our experience. Observation of two independent flowering events in culture amongst 42 non-induced transformation events is therefore highly significant. Dissection of these reproductive structures indicated fully formed ovules with silks were formed (FIG. 3). This early flowering is due to leaky FT expression in these particular transgenic events as estradiol inducer was not applied to these cultures. The other independent transgenic plants did not have reproductive structures and were transplanted to soil. This result demonstrates low level expression of non-native FT in corn plants is sufficient to induce very early flowering in plants having as few as three to five leaves. The inducible system for expressing FT allows the timing of this flowering to be controlled.

Example 5. FT Induction Using the AlcR Alcohol Inducible System

[0049] A plasmid construct of the basic design of the modified XVE system of Example 4, except the AlcR chimeric transcription factor is substituted for the modified XVE, is used. The A1cR operators are substituted for the LexA operators to have constructs similar to the AlcR system as described (U.S. Pat. No. 6,605,754 and Roslan et al., The Plant Journal (2001) 28(2), 225-235). The promoter to express the AlcR chimeric transcription factor is the full length inducible rice OsSUT1 promoter (U.S. Pat. No. 7,186,821), a phloem specific promoter. Additional suitable maize active phloem promoters included but are not limited to the group consisting of rice Rpp16 and Rpp17 (Asano et al., Plant Cell Physiol. 2002 June; 43(6):668-74); rice OsABCC1; Arabidopsis AtSUC2; and Arabidopsis AtPP2-A1 (accession no. At4g19840).

[0050] Transgenic regenerable calli are obtained to produce transgenic plants in soil. These transgenic plants are root drenched with a 2% solution of alcohol to induce FT expression. The induced plants flower between the five leaf and 15.sup.th leaf of development, depending on how early and often an alcohol drench is applied to the roots of the young plants.

Example 6. Inducible Expression of FT in Maize Causes Early Flowering in Maize Plants

[0051] Transgenic plants from embryogenic callus and young plants from Example 4 were grown to maturity in the greenhouse to obtain T1 or T2 seeds to test for early flowering. T1 or T2 seeds were germinated in the presence or absence of estradiol or diethylstilbestrol (DES) to induce FT expression in a beakers with wet paper towels to induce FT expression. One week old seedlings were transplanted to soil and sprayed with induced on alternate days for another week. Plants were then maintained normally in the greenhouse or indoor growth room and observed for flowering phenotypes. Depending on the transgenic line, non-induced plants were normal, had slightly early flowering, or some leaky expressors flowered in 3 to 4 weeks after germination.

[0052] Three independently transformed plant lines that had normal to slightly early flowering times when not induced were chosen for more detailed examination. These lines flowered in 3 to 4 weeks when induced, and had normal to early flowering times when not induced. Ovule (silk) production on the tassel structure (tassel seed phenotype) was observed first as early as three weeks on induced plants. The earliest flowering plants were pollinated 27 days after imbibing seeds in the presence of inducer (estradiol or DES) and had large well developed kernels by 39 days post seed imbibition. These seeds matured and were viable when germinated, demonstrating these early flowering plants were fertile and produced viable seed at much earlier times than control plants. Anther and pollen development were not quite as fast, with the first viable pollen appearing 38 days post germination. Control plants of the same genotype flowered in about 55 to 60 days in this experiment. This system has clear benefits for accelerated generation times. We note early flowering plants were obtained from either low constitutive levels of FT expression or when induced, as both types of constitutive (non-induced) or inducible expression plants were recovered in independent transformation events.