Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8233—Female-specific, e.g. pistil, ovule
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

• Heterosis also termed hybrid vigor, refers to the higher performance of a hybrid progeny in comparison to those of its parents.
• Several underlying mechanisms (dominance, overdominance, and epistasis) have been proposed to explain the genetic and molecular bases of heterosis 1 .
• Heterosis has been harnessed in crops, notably through the development of Fl hybrids in seed crops that exhibit superior yield potential and stability.
• Fl hybrid seeds must be renewed at every crop season because F2 progeny seeds are prone to trait segregation and lower global plant performance.
• a potentially revolutionary alternative to male sterility systems is the propagation of Fl seeds over generations in an immortalized manner through an asexual, clonal mode of reproduction called apomixis.
• Apomixis occurs naturally in more than 120 genera of angiosperms, notably in some wild relatives of crops such as maize, wheat, and millet 3 .
• attempts to find naturally occurring apomixis in crops or to transfer the genetically characterized apomictic loci from wild relatives to crops have so far failed 4 .
• meiosis is converted to mitosis through a set of three mutations (called MiMe for Mitosis instead of Meiosis 8,9 ) that target the three features that differentiate meiosis from mitosis:
• recombination and pairing are suppressed through the inactivation of a member of the recombination initiation complex (e.g. SPO11-1 10 or PA1R1 11 ).
• the joint migration of sister chromatids at meiosis is replaced by their separation through the inactivation of the cohesin REC8 12 .
• the second meiotic division is omitted through the inactivation of the cell cycle regulator OSD1 13 .
• MiMe as a platform, three strategies have been used to induce unreduced and unrecombined egg cells to develop into diploid clonal embryos: i. crossing MiMe with a cenh3 mutant line expressing a CENH3- variant protein 14 that induces paternal genome elimination in the zygote 15 ; ii. inactivating the sperm cell-expressed phospholipase gene (NLD/MATL/PLA1) 6 in MiMe 11 that likely also contributes to paternal genome elimination 18 ; or iii. expressing a parthenogenetic trigger in the egg cell 19 .
• NLD/MATL/PLA1 sperm cell-expressed phospholipase gene
• the first strategy implemented in Arabidopsis thaliana, requires a crossing step and is, therefore, a non-autonomous system as it cannot be propagated by selffertilization.
• the NLD/MATL/PLA1 mutation was found to alter plant fertility in rice (the fertility of the pairl/osrec8/ososdl/osmatl quadruple mutant is 10% of that of the control MiMe), and the frequency of clonal seeds in the resulting apomictic plants remained low (6— 8%) 17 .
• methods of generating progeny of a subspecies plant cross comprises, providing Fl progeny of a subspecies plant cross; transforming tissue of the Fl progeny with a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype; selecting transformed cells from the tissue; regenerating tissue from the transformed cells to form a transformed plant; and collecting the selfed- seed from the regenerated plant.
• a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide
• the tissue is embryonic or somatic tissue.
• the fertility rate of plants from the selfed-seed is at least 20% (e.g., at least 25, 30, 40, 50%) higher than the fertility rate of Fl plants, measured by production of F2 progeny, from a natural subspecies cross, wherein the fertility rate is the percent of ovules that produce viable seeds.
• the fertility rate of plants from the selfed-seed is at least 80% (e.g., at least 90% or at least 95%), wherein the fertility rate is the percent of ovules that produce viable seeds.
• the subspecies are rice, barley, wheat or maize subspecies.
• the subspecies from the subspecies plant cross are indica and japonica, aus and japonica, or indica and aus.
• the first plant and the second plant are classified as different species.
• a first plant of the subspecies plant cross is a wild plant and a second plant of the subspecies plant cross is domesticated.
• the Babyboom protein is at least 70, 80, 85, 90, 95, 98 or 100% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) ATREC8 or an ortholog thereof, and (iii) ATSPO11 or an ortholog thereof.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) REC8 or an ortholog thereof, and (iii) PAIR1 or an ortholog thereof.
• a gRNA targeting i) OSD1 or an ortholog thereof, (ii) REC8 or an ortholog thereof, and (iii) PAIR1 or an ortholog thereof.
• selfed-seed produced by the method as described above or elsewhere herein, or clonal progeny thereof.
• a plant clone of an Fl progeny plant from a subspecies plant cross comprising a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype, wherein the plant clone produces clonal seed.
• a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein
• a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meio
• the Babyboom protein is at least 70, 80, 85, 90, 95, 98 or 100% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) ATREC8 or an ortholog thereof, and (iii) ATSPO11 or an ortholog thereof.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) REC8 or an ortholog thereof, and (iii) PAIR1 or an ortholog thereof.
• the plant is a rice, barley, wheat or maize plant.
• a method of generating a triploid fertile plant comprising, generating triploid embryonic tissue comprising a heterologous a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype; regenerating tissue from the embryonic tissue to form a triploid plant; and collecting selfed-seed from the regenerated triploid plant, wherein the selfed-seed is triploid.
• a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein
• the generating comprises crossing:
• a diploid parent plant comprising a heterologous a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype with
• a haploid parent plant comprising a heterologous a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype.
• a haploid parent plant comprising a heterologous a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis
• the Babyboom protein is at least 70, 80, 85, 90, 95, 98 or 100% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) ATREC8 or an ortholog thereof, and (iii) ATSPO11 or an ortholog thereof.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) REC8 or an ortholog thereof, and (iii) PAIR1 or an ortholog thereof.
• triploid selfed- seed produced by the methods described above or elsewhere herein, or clonal triploid progeny thereof.
• a triploid or higher ploidy fertile plant comprising a heterologous a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype, wherein the triploid or higher ploidy fertile plant produces selfed-seed that are triploid or higher ploidy, respectively.
• a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein
• a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and
• the Babyboom protein is at least 70, 80, 85, 90, 95, 98 or 100% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) ATREC8 or an ortholog thereof, and (iii) ATSPO11 or an ortholog thereof.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) 0SD1 or an ortholog thereof, (ii) REC8 or an ortholog thereof, and (iii) PAIR1 or an ortholog thereof.
• the method comprises transforming tissue of the plant with a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-cell expressing promoter operably linked to a polynucleotide encoding a Babyboom protein and (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype; selecting transformed cells from the tissue; regenerating tissue from the transformed cells to form a transformed plant; and collecting the selfed-seed from the regenerated plant, wherein the selfed-seed display a frequency of parthenogenesis of at least 80% (or 90% or 95%).
• the tissue is embryonic or somatic tissue.
• the plant is a rice, barley, wheat or maize plant.
• the Babyboom protein is at least 70, 80, 85, 90, 95, 98 or 100% identical to SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) ATREC8 or an ortholog thereof, and (iii) ATSPO11 or an ortholog thereof.
• the MiME expression cassettes comprise at least one expression cassette encoding a gRNA targeting (i) OSD1 or an ortholog thereof, (ii) REC8 or an ortholog thereof, and (iii) PAIR1 or an ortholog thereof.
• the plant is a hybrid plant.
• the plant is an Fl of a subspecies cross.
• the plant is a triploid or tetrapioid plant.
• Figure 1A-B Ploidy and genotype of progeny plants of transformation events harboring the T314 and T315 apomixis-inducing T-DNA constructs.
• 1A Schematic representation of the T-DNA constructs used to induce the triple MiMe mutation and the triggering of parthenogenesis, resulting in synthetic apomixis.
• T313 sgRNA MiMe T- DNA Middle T314 sgRNA MiMe_pAtECS:BBMl T-DNA Lower: T315 sgRNA_pOsECS:BBMl T-DNA: LB and RB : left and right borders of the T-DNA ;
• p35S promoter of the Cauliflower Mosaic Virus (CaMV); 35S: polyadenylation signal of CaMV ;
• hpt cat int hygromycin phosphotransferase II with castor bean catalase intron;
• ZmUbi promoter, first intron and first exon of the maize Ubiquitin 1 gene;
• “Os”Cas9 rice- optimized Cas9 coding sequence ;
• NLS nucleus localization signal fused to Cas9; OSD1, OSD1/2, PAIR1 and REC8: Four cassettes including sgRNAs (20 bp crRNA specific to the target gene + 82b
• IB Principle for formation of tetrapioid and diploid clonal progenies in MiMe and MiMe + BBM1 plants, respectively.
• C Representative flow cytometry histograms of DAPI-marked nuclei suspensions isolated from young leaf blade of a diploid (upper) and tetrapioid (lower) progeny plants.
• D upper: Genealogy of the plants selected for whole-genome sequencing including IF and D24 parents (two plants each); heterozygous Fl hybrid BRS-CIRAD 302 (two plants), six F2 sexual progenies; T314 15.1 and 37.7 primary transformants (TO); three T1 progeny plants of each of the sequenced TO plants; three T2 progenies of each of the sequenced T1 plants (i.e., nine T2 plants per event).
• Lower Graphical representation of genotypes of the 12 rice chromosomes established from whole genome sequences of homozygous parents, heterozygous Fl hybrid, F2 progeny plants, TO events T314 15.1 and 37.7 and their T1 and T2 progenies. Changes in color along F2 progeny chromosomes mark heterozygous-to-homozygous transitions resulting from meiotic crossovers.
• FIG. 2A-F Phenotype, panicle fertility, and grain quality of progeny plants of selected apomictic events harboring the T314 and T315 T-DNA constructs.
• 2A Phenotypes of plants grown under controlled greenhouse conditions: Left: Five F2 progeny plants derived from the self-fertilization of BRS-CIRAD 302 compared to a BRS-CIRAD 302 Fl plant. Right: Three T1 progenies from T314 15.1 event compared to a BRS-CIRAD 302 Fl plant.
• 2B Phenotypes of T2 progenies grown under controlled greenhouse conditions: 5-6 T2 progeny plants of a T1 plant of events T314 15.1, T31437.7, T315 5.4 and T315 8.1 are compared to a BRS-CIRAD 302 Fl plant. Senescent leaves of the plants have been removed for photographing.
• 2C Panicles of the BRS-CIRAD 302 Fl hybrid and of T314 15.1 T2 plants. The master panicles of five distinct plants have been pooled for photographing.
• 2D Distribution of seed filling rate among BRS-CIRAD 302 Fl plants, and T2 progeny plants of T314 events (15.1 and 37.7) and T315 events (5.4 and 8.1 events).
• Average panicle fertilities of the apomictic lines range from 60 to 80% of those of the control plants. Significance of the differences are based on Duncan’s test.
• 2E Husked and dehulled seeds of IF and D24 parents, Fl and F2 generations and apomictic lines. Upper: IF and D24 parents, Fl hybrid seeds harvested on IF parent, F2 seeds harvested on the Fl hybrid. Lower: T3 seeds harvested from apomictic plants in the four selected apomictic lines.
• Figure 3A-B Figure 3 A. Synthetic apomixis construct used to transform Kalingalll-Kitaake (KKIII) indica-japonica hybrids. This is the same construct that was used for transformation of hybrid BRS-CIRAD 302.
• Figure 3B Photos of panicles from three independent KKIII transformants. There is variation between the events, with the left and middle plants showing high fertility, whereas the plant on the right has fertility similar to that observed with untransformed K-KIII hybrids.
• the middle plant (line KK32.3), displayed a balance of an acceptable fertility of 87 % and a moderate parthenogenesis frequency of 60%.
• the KK-III hybrid plants with the synthetic apomixis trait were generated using the same protocols as the BRS-CIRAD 302 hybrids described in Figures 1, and 2, and detailed in the Methods section of Example 1.
• An "endogenous” or “native” gene or protein sequence refers to a gene or protein sequence that is naturally occurring in the genome of the organism.
• a polynucleotide or polypeptide sequence is "heterologous" to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form.
• a promoter when a promoter is said to be operably linked to a heterologous coding sequence, it means that the coding sequence is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).
• a "plant promoter” is a promoter capable of initiating transcription in plant cells.
• a “constitutive promoter” is one that is capable of initiating transcription in nearly all tissue types, whereas a “tissue-specific promoter” initiates transcription only in one or a few particular tissue types.
• operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• a nucleic acid expression control sequence such as a promoter, or array of transcription factor binding sites
• plant includes whole plants, shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, plant tissue (e.g., vascular tissue, ground tissue, and the like), cells (e.g., guard cells, egg cells, trichomes and the like), and progeny of same.
• shoot vegetative organs and/or structures e.g., leaves, stems and tubers
• roots e.g., bracts, sepals, petals, stamens, carpels, anthers
• ovules including egg and central cells
• seed including zygote, embryo, endosperm, and seed coat
• fruit e.g., the mature
• the class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid, and hemizygous.
• a "transgene” is used as the term is understood in the art and refers to a heterologous nucleic acid introduced into a cell by human molecular manipulation of the cell's genome (e.g., by molecular transformation).
• a "transgenic plant” is a plant that carries a transgene, i.e., is a genetically-modified plant.
• the transgenic plant can be the initial plant into which the transgene was introduced as well as progeny thereof whose genomes contain the transgene.
• nucleic acid or “polynucleotide sequence” refers to a single or doublestranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase, and/or formation of double-stranded duplexes, and do not significantly alter expression of a polypeptide encoded by that nucleic acid.
• nucleic acid sequence encoding refers to a nucleic acid which directs the expression of a specific protein or peptide.
• the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein.
• the nucleic acid sequences include both the full length nucleic acid sequences as well as non-full length sequences derived from the full length sequences. It should be further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
• nucleic acid sequences or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• Two nucleic acid sequences or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below.
• sequence identity When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity.
• a conservative substitution is given a score between zero and 1.
• the scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California, USA).
• substantially identical used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence (e.g., any of SEQ ID NOs: 1-59). Alternatively, percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below.
• sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
• sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Methods of alignment of sequences for comparison are well-known in the art.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
• T is referred to as the neighborhood word score threshold (Altschul et al, supra).
• These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
• the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
• Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score.
• Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10’ 5 , and most preferably less than about 10' 20 .
• An "expression cassette” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively.
• a single nucleic acid construct comprising (i) a first expression cassette comprising an egg-specific promoter operably-linked to a polynucleotide encoding a Babyboom polypeptide, (ii) a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and (iii) sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype sufficient expression cassettes to induce a meiosis-to- mitosis (Mime) phenotype greatly improves efficiency of generation of plants that produce clonal seed.
• a first expression cassette comprising an egg-specific promoter operably-linked to a polynucleotide encoding a Babyboom polypeptide
• a second expression cassette comprising a promoter operable linked to an RNA-guided nuclease and
• the single nucleic acid construct described herein comprise an expression cassette for the expression of a Babyboom polypeptide, an RNA-guided nuclease, and sufficient gRNAs to generate the Mime phenotype, and optionally an expression cassette expressing a selectable (e.g., antibiotic or herbicide resistance) marker.
• a selectable e.g., antibiotic or herbicide resistance
• BABYBOOM polypeptides Any naturally-or non-naturally-occurring active BABYBOOM polypeptide from a sexually reproducing plant can be expressed as described herein so long as the polypeptide (and/or RNA encoding the polypeptide) is expressed in egg cells in the plant.
• BABY BOOM polypeptides contain two conserved AP2 domains. The corresponding transcripts lack a miR172 binding site, thereby distinguishing BABY BOOM polypeptides from many other AP2 domain proteins that contain a miR172 binding site.
• the BABYBOOM polypeptide is from a species of plant of the genus Abelmoschus, Allium, Apium, Amaranthus, Arachis, Arabidopsis, Asparagus, Atropa, Avena, Benincasa, Beta, Brassica, Cannabis, Capsella, Cica, Cichorium, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Cynasa, Daucus, Diplotaxis, Dioscorea, Elais, Eruca, Foeniculum, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomea, Lactuca, Lagenaria, Lepidium, Linum, Lolium, Luffa, Luzula, Lycopersicon, Malus, Manihot, Majorana, Medicago, Momodica, Musa, Nicotian
• the BABYBOOM polypeptide is identical or substantially identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. See, also, Chahal, et al., Front. Plant Sci., 14 July 2022.
• the plant comprises heterologous expression cassette comprising a promoter that at least directs expression to egg cells operably linked to a BABYBOOM polypeptide as described herein.
• the promoter is egg cell-specific, meaning the promoter drives expression only or primarily in egg cells. “Primarily” means that if there is expression in other tissue the levels are no more than 1/10 of the expression levels in egg cells as measured by quantitative RT-PCR.
• Exemplary promoters that drive expression in at least egg cells of a plant include, but are not limited to, the promoter of the egg-cell specific gene EC 1.1 (e.g., SEQ I D NO:23), EC1.2, EC1.3, EC1.4, or EC1.5. See, e.g. Sprunck et al. Science, 338:1093-1097 (2012); AT2G21740; Steffen et al., Plant Journal 51: 281-292 (2007).
• the rice-specific promoter comprises SEQ ID NO:22, i.e., the rice egg cellspecific promoter sequence from the LOC_Os03g 18530 OsECAl gene.
• the Arabidopsis DD45 promoter is used to express in rice egg cell (Ohnishi et al. Plant Physiology 165: 1533-1543 (2014).
• An exemplary DD45 promoter sequence can comprise, for example, SEQ ID NO:21.
• Other promoters that can be used for egg cell expression include promoters of the egg cell-specific ECS1 (SEQ ID NO:60) and ECS2 (SEQ ID NO:61) genes (Yu et al., 2021 Nature 592:433-437) and the RWD2 gene (Koszegi et al. 2011 The Plant Journal 67:280-291).
• the expression cassette further comprises a transcriptional terminator.
• exemplary terminators can include, but are not limited to, the rbcS E9 or nos terminators.
• the expression cassette will include an egg cell enhancer.
• Exemplary egg cell enhancers include, but are not limited to, the EC1.2 enhancer or EASE enhancer (Yang et al., Plant Physiol. 139:1421-32 (2005).
• the single construct will also comprise an expression cassette comprising a promoter operably linked to an RNA-guided nuclease.
• the RNA-guided nuclease can recognize a sequence of a target nucleic acid e.g., via an RNA guide), bind to the target nucleic acid, and modify the target nucleic acid.
• the RNA-guided nuclease has nuclease activity.
• the RNA-guided nuclease can modify the target nucleic acid by cleaving the target nucleic acid.
• the introduction of inserts or deletions by the error-prone non-homologous end joining repair of double-strand breaks (DSBs) introduces frame-shift mutations and for example subsequent premature stop codons, leading to mRNA elimination by nonsense-mediated mRNA decay.
• the Cas nuclease can direct cleavage of one or both strands at a location in a target nucleic acid.
• Cas nucleases include Casl, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, homologs thereof, variants thereof, mutants thereof, and derivatives thereof.
• Type II Cas nucleases There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66).
• Type II Cas nucleases include Casl, Cas2, Csn2, Cas9, and Cfpl. These Cas nucleases are known to those skilled in the art.
• the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP_269215
• the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470.
• Cas nucleases can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Myco
• Torquens llyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifr actor salsuginis, Sphaerochaeta globus, Fibrobacter succino genes subsp.
• Jejuni Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.
• Cas9 protein refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active.
• the Cas9 enzyme can comprise one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifr actor, and Campylobacter.
• the Cas9 can be a fusion protein, e.g., the two catalytic domains are derived from different bacteria species.
• a Cas protein can be a Cas protein variant.
• useful variants of the Cas9 nuclease can include a single inactive catalytic domain, such as a RuvC’ or HNH’ enzyme or a nickase.
• a Cas9 nickase has only one active functional domain and can cut only one strand of the target DNA, thereby creating a single strand break or nick.
• the Cas9 nuclease can be a mutant Cas9 nuclease having one or more amino acid mutations.
• the mutant Cas9 having at least a D10A mutation is a Cas9 nickase.
• the mutant Cas9 nuclease having at least a H840A mutation is a Cas9 nickase.
• Other examples of mutations present in a Cas9 nickase include, without limitation, N854A and N863A.
• a double-strand break can be introduced using a Cas9 nickase if at least two DNA-targeting RNAs that target opposite DNA strands are used.
• a double-nicked induced double-strand break can be repaired by NHEJ or HDR (Ran et al., 2013, Cell, 154:1380-1389).
• Non-limiting examples of Cas9 nucleases or nickases are described in, for example, U.S. Patent No.
• the Cas9 nuclease or nickase can be codon-optimized for the target cell or target organism.
• the Cas nuclease can be a high-fidelity or enhanced specificity Cas9 polypeptide variant with reduced off-target effects and robust on-target cleavage.
• Cas9 polypeptide variants with improved on-target specificity include the SpCas9 (K855A), SpCas9 (K810A/K1003A/R1060A) (also referred to as eSpCas9(1.0)), and SpCas9 (K848A/K1003A/R1060A) (also referred to as eSpCas9(l.l)) variants described in Slaymaker et al., Science, 351(6268):84-8 (2016), and the SpCas9 variants described in Kleinstiver et al., Nature, 529(7587):490-5 (2016) containing one, two, three, or four of the following mutations: N497A, R661A, Q695A,
• the promoter operably linked to the sequence encoding the RNA guided nuclease can be a constitutive promoter or an egg-specific promoter or be otherwise selected such that the RNA guided nuclease is expressed in egg cells.
• constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, the parsley UBI promoter (Kawalleck et al., Plant Mol Biol. (1993 Feb) 21(4):673-84), RPS5 (Hiroki Tsutsui et al.
• each expression cassette in the single construct uses a different promoter.
• RNA-guided nuclease will be expressed with a sufficient set of expression cassettes directing expression of guide RNAs (gRNAs) to induce a meiosis-to-mitosis phenotype.
• gRNAs guide RNAs
• Plant genes to be targeted to obtain a MiMe phenotype are known and are also described below.
• expression of a single guide RNA per gene can be sufficient to reduce expression of each target gene, but if desired, two or more guide RNA can be targeted to one of more of the genes to further reduce its expression.
• a guide RNA (gRNA) sequence is a sequence that interacts with a site- specific or targeted nuclease and specifically binds to or hybridizes to a target nucleic acid within the genome of a cell, such that the gRNA and the targeted nuclease colocalize to the target nucleic acid in the genome of the cell.
• Each gRNA includes a DNA targeting sequence or protospacer sequence of about 10 to 50 nucleotides in length that specifically binds to or hybridizes to a target DNA sequence in the genome.
• the DNA targeting sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
• the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence.
• the gRNA does not comprise a tracrRNA sequence.
• the guide sequence can be used in a single-guide RNA (sgRNA) as described below, or in a split crRNA + tracrRNA construct.
• the targeted nuclease (e.g., a Cas protein) is guided to its target DNA by a single-guide RNA (sgRNA).
• sgRNA is a version of the naturally occurring two-piece guide RNA (crRNA and tracrRNA) engineered into a single, continuous sequence.
• An sgRNA typically contains (1) a guide sequence (e.g., the crRNA equivalent portion of the sgRNA) that targets the Cas protein to the target DNA, and (2) a scaffold sequence that interacts with a nuclease such as a Cas protein (e.g., the tracrRNAs equivalent portion of the sgRNA).
• An sgRNA may be selected using a software.
• considerations for selecting an sgRNA can include, e.g., the PAM sequence for the Cas9 protein to be used, and strategies for minimizing off-target modifications.
• Tools such as NUPACK® and the CRISPR Design Tool can provide sequences for preparing the sgRNA, for assessing target modification efficiency, and/or assessing cleavage at off-target sites.
• the guide sequence in the sgRNA may be complementary to a specific sequence within a target DNA.
• the 3’ end of the target DNA sequence can be followed by a PAM sequence.
• Approximately 20 nucleotides upstream of the PAM sequence is the target DNA.
• a Cas9 protein or a variant thereof cleaves about three nucleotides upstream of the PAM sequence.
• the guide sequence in the sgRNA can be complementary to either strand of the target DNA.
• the promoter operably linked to the sequence encoding the guide RNA can be a constitutive promoter or an egg- specific promoter or be otherwise selected such that the guide RNA is expressed in egg cells.
• constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens, the parsley UBI promoter (Kawalleck et al., Plant Mol Biol. (1993 Feb) 21(4):673-84), RPS5 (Hiroki Tsutsui et al. Plant and Cell Physiology (2016)); 2X35S (Belhaj, Khaoula, et al. Plant methods 9.1 (2013): 39); AtUBIlO (Callis J, et al.
• CaMV cauliflower mosaic virus
• 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumafaciens the parsley UBI promoter
• RPS5 Hiroki Tsutsui et al. Plant and Cell Physiology (2016)
• 2X35S Belhaj, Khaoula, et al. Plant methods 9.1
• a plant having the MiMe (mitosis instead of meiosis) genotype is a plant in which a deregulation of meiosis results in a mitotic-like division and in which meiosis is replaced by mitosis.
• Plants having the MiMe genotype produce functional (e.g., diploid) gametes that are genetically identical to their parent.
• Exemplary MiMe plants combine phenotypes of (1) no second meiotic division, (2) no recombination and (3) modified chromatid segregation.
• MiMe plants are exemplified by MiMe-1 plants as described by d'Erfurth, I. et al. PLoS Biol 7, el000124 (2009) and WO2001/079432) and MiMe-2 plants as described by d'Erfurth, I. et al. PLoS Genet 6, el000989 (2010).
• the MiMe phenotype is induced by inhibiting or mutating OSD1 or an ortholog thereof, REC8 or an ortholog thereof, and at least one of SPO11 or PRD1, or PRD2 or PRD3/PAIR1 (see, e.g., Mieulet D consume Cell Res. 2016 Nov; 26(11): 1242-1254).
• Exemplary MiMe- 1 plants combine inactivation of the OSD 1 gene, with the inactivation of two or more other genes, one which encodes a protein necessary for efficient meiotic recombination in plants (e.g., SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1), and whose inhibition eliminates recombination and pairing (see, e.g., Grelon M, et al.
• Exemplary MiMe-2 plants combine inactivation of the TAM gene with the inactivation of two or more other genes, one which encodes a protein necessary for efficient meiotic recombination in plants (e.g., SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1), and whose inhibition eliminates recombination and pairing, and another which encodes a protein necessary for the monopolar orientation of the kinetochores during meiosis, e.g., REC8, and whose inhibition modifies chromatid segregation.
• a protein necessary for efficient meiotic recombination in plants e.g., SPO11-1, SPO11-2, PRD1, PRD2, or PAIR1
• REC8 e.g., REC8
• OSD1 gene sequences include, e.g., those described in US Patent Publication No. 2014/0298507 and rice and Arabidopsis OSD1 protein sequences as provided in SEQ ID NOS:25 and 27, respectively.
• Exemplary TAM gene sequences are described in, e.g., US Patent Publication No. 2014/0298507.
• Arabidopsis TAM1 protein sequence is provided as SEQ ID NO:37.
• Illustrative rice Cyclin-Al protein sequences are provided as SEQ ID NOS:39, 41, 43, 45, and 47.
• Illustrative Cyclin-A3 protein sequences are provided as SEQ ID NOS:49 and 51.
• Exemplary Arabidopsis DYAD cDNA coding sequence and the sequence of the protein encoded by the nucleic acid are provided as SEQ ID NOS:70 and 71, respectively.
• Exemplary rice DYAD homolog (SWITCH 1) protein sequences are provide as SEQ ID NOS:55, 57, and 59.
• SPO11-1 and SPO11-2 proteins are provided in US Patent Publication No. 2014/0298507.
• An illustrative Arabidopsis SPO11-2 protein sequence is provided as SEQ ID NO:31.
• Arabidopsis PAIR1 is described in, e.g., US Patent Publication No. 2014/0298507.
• An exemplary rice PAIR1 protein sequence is provided as SEQ ID NO:29.
• Exemplary rice and Arabidopsis REC8 protein sequences are provided as SEQ ID NOS:51 and 53, respectively.
• sufficient expression cassettes to produce the MiMe phenotype include at least one expression cassette comprising a promoter operably linked to one or more guide RNA targeting a gene or coding sequence encoding (a) a TAM (Cylin A CYCA1;2) or DYAD protein or ortholog thereof; (b) a protein involved in initiation of meiotic recombination in plants exemplified herein as SPO11-1 ; SPO11-2; PRD; PRD2; or PAIR1 (also called PRD3) or ortholog thereof; and (c) a protein necessary for the monopolar orientation of the kinetochores during meiosis for example REC8 protein or ortholog thereof.
• Orthologs have the functionality of the proteins described herein but are from different plant species. Orthologs can be substantially identical to the polypeptides as provide herein or can otherwise be selected from genomic databases.
• sufficient expression cassettes to produce the MiMe phenotype include at least one expression cassette comprising a promoter operably linked to one or more guide RNA targeting a gene or coding sequence encoding (a) an OSD 1 protein or ortholog thereof; (b) a protein involved in initiation of meiotic recombination in plants exemplified herein as SPO11-1; SPO11-2; PRD; PRD2; or PAIR1 (also called PRD3) or ortholog thereof; and (c) a protein necessary for the monopolar orientation of the kinetochores during meiosis, for example REC8 proteinor ortholog thereof.
• the above-described expression cassettes are delivered on a single nucleic acid (e.g., DNA) construct, thereby allowing for highly efficient induction of parthenogenesis.
• the methods are not limited to a particular method of transformation, in some embodiments, the single construct is delivered by Agrobacterium as a T-DNA.
• the single DNA construct compromising the expression cassettes as described herein can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
• the virulence functions of the Agrobacterium tumefaciens host will direct the transfer of the T-DNA into plant cells when the cell is infected by the bacteria.
• Agrobacterium tumefaciens-me iated transformation techniques including disarming and use of binary vectors, are well described in the scientific literature. See, for example, Horsch et al. Science 233:496-498 (1984), and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803 (1983).
• transformation will be performed on embryonic plant tissue.
• Agrobacterium tumefaciens can be co-cultivated with seed embryo-derived secondary calluses (see, e.g., Sallaud, C. et al., Theor. Appl. Genet. 106, 1396-1408 (2003); US Patent No. 10,584,345; EP0290395; and US2011/0212525).
• transformation will be performed on somatic tissue.
• transformation will be performed on plant protoplasts.
• transformation will be performed on immature leaves, inflorescences, pollen or other regenerable tissue.
• Transformed cells can subsequently be selected (e.g., selecting for antibiotic resistance or other selectable marker introduced with the T-DNA or as otherwise known in the art).
• Primary transformed cells can subsequently be regenerated into plants.
• the plant manipulated as described herein can be any plant species.
• the plant is a dicot plant.
• the plant is a monocot plant.
• the plant is a grass.
• the plant is a cereal (e.g., including but not limited to Poaceae, e.g., rice, barley, wheat, maize).
• the plant is a species of plant of the genus Abelmoschus, Allium, Apium, Amaranthus, Arachis, Arabidopsis, Asparagus, Atropa, Avena, Benincasa, Beta, Brassica, Cannabis, Capsella, Cica, Cichorium, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Cynasa, Daucus, Diplotaxis, Dioscorea, Elais, Eruca, Foeniculum, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomea, Lactuca, Lagenaria, Lepidium, Linum, Lolium, Luffa, Luzula, Lycopersicon, Malus, Manihot, Majorana, Medicago, Momodica, Musa, Nicotiana, Olea, Ory
• the plant is a rice, barley, wheat, maize, sorghum, millet, oat, triticale, rye, fonio, or other cereal plant.
• the plant transformed is an Fl embryo is a hybrid resulting from a cross of two in-bred plant lines.
• plant hybrids are offspring of parents that differ in genetically determined traits.
• the Fl embryo is heterozygous, i.e. differing in nucleotide sequence between the parental alleles, in at least 10, 20, 30, 40, 50, 60, 70, 80% or more genes.
• the efficiency of parthenogenesis of plants generated by the methods described herein are at least 80%, 85%, 90% or 95%, meaning that at least this percentage of progeny of a plant generated by the methods will be clonal, i.e., genetically the same.
• Example 2 while the methods are applicable to any plant, the methods can be used to generate agronomically viable seed from FIs resulting from crosses of diverse subspecies that would generally result in FIs having low or rare fertility.
• Exemplary subspecies that can be crossed to form an Fl embryo that is subsequently transformed by the single construct described herein can include, for example, rice subspecies indica and japonica and aus.
• the Fl is the result of a cross between indica and japonica, aus and japonica, or indica and aus.
• the Fl is the result of a cross between Common wheat (Triticum spp.) with rye (Secale cereale L.). This cross produces Triticale, which is a desirable crop due to its general vigor, seed quality and hardiness.
• the cross is between a wild and a domesticated plant.
• Oryza sativa is a cultivated species and its wild relative is Oryza rufipogon.
• African cultivated rice is Oryza glaberrima and its wild relative is Oryza barthii.
• the Fl embryo is from a cross between Oryza sativa and Oryza rufipogon or between Oryza glaberrima and Oryza barthii.
• the cross is between two different species that naturally produce infertile or inviable Fl progeny. Examples can include but are not limited to crosses between wheat and rye to make triticale.
• the methods described herein can also be used to generate triploid or tetrapioid or plants of higher ploidy that will have the capacity to reproduce, by forming clonal seeds.
• sugarcane hybrids give rise to complex higher polyploids, that currently can only be propagated by cuttings.
• a cross between a diploid and haploid plant, or two diploids, or a haploid and a triploid, or other ploidy combinations (for example but not limited to odd number ploidy such as 5, 7, 9, etc.) that would normally result in inviable or infertile progeny, can now be used to generate plants of any ploidy that produce clonal seeds of the same ploidy.
• the diploid parent will produce diploid unreduced gametes and the haploid plant will produce haploid unreduced gametes.
• the seeds from the cross will abort.
• embryo rescue can be performed from the immature seeds and cultured to form plants, using standard protocols for embryo rescue in plants.
• the regenerated plants will be triploid.
• the triploid plants will comprise the expression cassettes for egg expression of Babyboom and exhibit the MiMe phenotype, meaning that the triploid will produce clonal seed without going through meiosis. Accordingly, triploid plants that produce clonal seed, and comprising the expression cassettes as describe herein, are provided.
• the methods above can be applied to generate fertile triploid or plants of higher ploidy from any species of plant.
• the plant can be for example a tuber crop plant (e.g., potato, cassava, yam, or other tubers), allowing for distribution of the plants by seed rather than as currently occurs (i.e., via tubers).
• T314 (15.1 and 37.7) and four T315 (3.2, 5.4, 8.1, and 8.2) events, which produced diploid progeny (Tl) at a rate of more than 92%.
• T2 progeny plants n > 40
• T315 3.2, 5.4, 8.1, and 8.2 events
• BRS-CIRAD 302 anthers In our greenhouse conditions, BRS-CIRAD 302 anthers exhibited average pollen viability of 60%. Nevertheless, pollen viability that is reduced by half is considered sufficient for full panicle fertilization in rice, as illustrated in commercial Fl hybrids carrying the rice Hong Liang (HL) and Boro II (BT) cms systems, which have a gametophytic mode of male fertility restoration 2 . As for the T2 clonal progenies of T314 and T315 events, they also showed reduced panicle fertilities, with average grain filling rates falling within a narrow 27%-35.5 % range (Table 3; Figure 2C and D). Thus, although fertilities were reduced in both BRS- CIRAD 302 control plants and apomictic progenies in greenhouse conditions, apomicts showed further reductions in fertility compared to the BRS-CIRAD 302 control plants (Table 3).
• ovules formed dyads (as verified by clearing and callose deposition patterns on meiotic cell walls), followed by degradation of the micropylar spore and selection of the chalazal functional megaspore, resulting in gametogenesis.
• a range of low-frequency abnormalities were also observed, such as tetrads, triads, and abortive or persisting dyads, which might result in abnormal gametogenesis that could partly explain the reduced female fertility.
• imbalance in chromosome segregation may occur at the first division leading to unbalanced 2n spores/gametophytes with reduced viability.
• T314 and T315 Kitaake plants with high-frequency (84%) apomixis we next examined the seed set of T1 plants from these lines. While wild-type Kitaake plants exhibited nearly 90% fertility, panicle fertility of apomictic lines reached 74% in a small number of transgenic lines examined. Thus, although apomicts exhibited roughly 16% less fertility compared to wild type, events with high- frequency apomixis and reasonably high (but not yet complete) fertility can be generated in a different genetic background.
• MiMe endosperm is expected to be initially hexapioid instead of triploid as it results from the fusion of the two diploid central cell nuclei and a diploid pollen sperm nucleus.
• a related and more specific conclusion is that the requirement for a fertilized endosperm for viable seeds does not set an upper limit for the frequency of parthenogenesis.
• the necessity for an endosperm could have prevented high-efficiency clonal seed formation because of a potential conflict between parthenogenetic zygote formation and central cell fertilization that triggers endosperm development.
• the fertilization of the central cell requires the Egg Cell 1 (ECI) protein, which is secreted exclusively by the egg cell and necessary for inducing the two pollen-delivered sperm cells to fuse with both the egg cell and the central cell 30 .
• ECI Egg Cell 1
• the BRS-CIRAD 302 control plants exhibited incomplete and highly variable panicle fertility that ranged from 25 to 65% under greenhouse conditions. Pollen viability in the hybrid, which was on average 60%, can probably not explain the defective seed setting rate in the Fl hybrid plants. Also, in the apomictic lines, higher pollen viability (80-90%) was observed without being associated with an improved seed set. A tentative cause of the reduced panicle fertility of the hybrid could be a detrimental interaction of the WA cytoplasm with the greenhouse environment through pleiotropic effects affecting other traits involved in fertilization. The apomictic lines were not fully fertile either under our greenhouse conditions and exhibited an average grain filling rate ranging from 27 to 35% across the lines, representing 60-80% of that of the control Fl hybrid.
• the first is a potential improvement in grain quality, which has long been a limiting factor in Fl hybrids; this has restricted their adoption and has been partly ascribed to the segregating F2 genotype of harvested seed endosperms. In synthetic apomicts, all the endosperms share the same Fl genotype, and this should result in more predictable grain quality features.
• a second advantage is avoiding the use of a single cytoplasm (e.g., WA cytoplasm) over large acreages of cultivation, which may make Fl hybrids more susceptible to disease outbreak 43 or reliant on very specific environments for environmental male sterility seed production systems.
• a third advantage is that it could widen the breadth of tested hybrid combinations that have so far been restricted by the long and tedious preliminary process of converting the parental lines to cms prior to multisite evaluation.
• Progress in understanding and harnessing the dispensable genome which has been demonstrated to harbor a wealth of adaptation genes 44 , may allow a more informed choice of genome combinations for developing future climate-smart apomictic hybrids. Having an efficient tool for converting hybrids to apomixis will be very valuable in this respect. Beyond the well reported yield performance and stability qualities, apomixis should therefore allow breeders to harness the potential of Fl hybrids that exhibit biotic and abiotic stress tolerance and are thus better equipped to deal with the challenges posed by global climate change and increased food demand.
• Hybrids are agriculturally important because they can greatly outperform standard varieties through a phenomenon called heterosis or hybrid vigour.
• hybrids In rice, hybrids have been used to increase yields by 25% to 50%.
• under low nitrogen indica/japonica hybrid rice has significantly higher yields than japonica/japonica hybrid rice (Chu et al. 2019, Field Crops Research, Volume 243, 107625, ISSN 0378-4290).
• this reproductive isolation barrier can be overcome by using a methods described herein, resulting in synthetic apomixis.
• a method in rice, called synthetic apomixis that results in clonal seed formation at frequencies up to 29% (Khanday et al. 2019 Nature) has been described previously.
• This technique can be used for stable propagation of hybrids without genetic segregation. It involves substitution of mitosis for meiosis in the germline (MiMe), followed by parthenogenesis induced by expressing a transcription factor BBM1 in the egg cell.
• KKIII hybrids produced viable clonal diploid progeny through parthenogenesis, at frequencies ranging from 4% to 100%.
• At least one line KK32.3 displayed a balance of an acceptable fertility of 87% and moderate parthenogenesis frequency of 60%. It is expected that with more events transformants that have higher parthenogenesis frequencies in addition to being fertile can be obtained by these same methods.
• This method can be applied to inter- sub species hybrids arising from different combinations of hundreds of indica and japonica genotypes, greatly expanding the availability of hybrid rice by exploiting a new landscape of genetic diversity and heterosis that is currently inaccessible.
• the method can also applied to other crop plants, in situations where desirable hybrid combinations cannot be cultivated because of sterility arising from pre-zygotic reproductive isolation.
• Viable zygotes resulting from intergeneric and interspecific hybridization in plants that carry parental chromosomes from both species will produce infertile flowers due to abnormal segregation of parental chromosomes at meiosis (e.g., crosses between wheat and rye, used for making triticale). Skipping meiosis of the intergeneric/interspecific hybrid through apomixis should allow novel combinations of genomes, and propagate novel genome associations through seeds.
• the method can also be used to propagate other examples of high performing genetic combinations that have vigorous vegetative growth but poor fertility. For example, triploid plants are more vigorous than diploid plants, but they have high sterility due to failure of gamete formation after meiosis. Because the method eliminates meiosis, viable gametes can be formed even from triploids. This is followed by parthenogenesis using the synthetic apomixis construct, ensuring that the progeny will also be triploid. This step is necessary to avoid the doubling the chromosome number in every generation, that would otherwise occur with sexual embryos after the elimination of meiosis.
• BRS-CIRAD 302 a CIRAD-EMBRAPA hybrid of rice released in 2010 in Brazil
• BRS-CIRAD 302 is a high-yielding indica/indica Fl hybrid resulting from crossing a male sterile line of IF (CIRAD 464), bearing the Wild Abortive (WA) cytoplasmic male sterility, and the D24 line carrying the restorer nuclear genes.
• Grains harvested on BRS-CIRAD 302 exhibit superior quality, according to Graham’s 45 classification, i.e., a long and slender shape, low breakage rate, and high amylose content.
• Transgenic plants TO, Tl, T2, and T3 generations
• parental lines IF and D24, BRS-CIRAD 302 hybrid, and F2 progenies were grown in containment greenhouse facilities under natural light supplemented by light provided by LEDs (12h:12h photoperiod) under 60% hygrometry and 28 °C day and 20 °C night temperatures.
• Wild- type and transgenic events (TO and Tl plants) of cv. Kitaake were grown at UC DAVIS under greenhouse conditions as previously described 19 .
• T-DNA construct preparation Three constructs were prepared ( Figure 1A): 1. the 4sgMiMe (T313) construct was created by inserting a 2,067 bp attBl-Attb2 fragment from the T-DNA of the pCAMBIA2300-MiMe 4sgRs vector 19 , containing four single guide RNA (sgRNA) cassettes each driven by an OsU6 promoter 46 , into a pDONR207 vector by Gateway (Clontech) cloning.
• sgRNA single guide RNA
• the 2G9 binary vector T-DNA region harbors a rice codon-optimized Cas9 coding region 47 driven by the maize ubiquitin promoter 48 , a selectable cassette containing the hpt gene with a catalase intron 49 , driven by the CaMV 35S promoter, and a cmR ccdb Gateway cloning site. 2.
• the sgMiMe_pAtECS:BBMl (T314) vector was prepared following blunt-end insertion of the 3,617 bp EcoR fragment of pCAMBIA1300-DD45:BBMl:Nos 19 containing the Arabidopsis EC1.2 egg cell-specific promoter driving the OsBABYBOOMl (OsBBMl) coding sequence terminated by the Nopaline synthase (Nos) polyadenylation signal, into the compatible Pme site of T313, situated proximal to the right border of the T-DNA. 3.
• the sgMiMe_pOsECS:BBMl (T315) vector was prepared by releasing the 2,828 bp Xbal-Asel fragment of the pCAMBIA1300_pOsECAl.l:OsBBMl:nosT plasmid, containing the promoter of the rice egg cell-specific OsECAl (LOC_Os03gl8530) gene 31,50 driving OsBBMl terminated by the Nos polyadenylation signal (Khanday and Sundaresan, unpublished), followed by its blunt-end insertion into the Pme cloning site of T313.
• OsBBMl cassettes adjacent to the T- DNA RB was ascertained in both T314 and T315 by sequencing.
• the sequences of the sgRNAs targeting rice PAIR1, REC8 and OSD1, the BBM1 coding region, and the Arabidopsis thaliana EC1.2 promoter region sequences were those originally detailed in refs. 21,30 .
• the binary vectors were introduced into Agrobacterium tumefaciens strain EHA105 by electroporation. The three constructs were cloned by the GENSCRIPT company (Leiden, the Netherlands).
• Transformation of BRS-CIRAD 302 was carried out by cocultivation of mature Fl seed embryo-derived secondary calluses with EHA 105 Agrobacterium cell suspensions, selection of transformed cell lines based on hygromycin tolerance, and primary transformant (TO) regeneration following the procedure detailed by Sallaud and co-workers 51 . Slight changes to the procedure include the reduction of Agrobacterium cell suspension OD to 0.01 and lengthening of some phases of selection due to slower growth of transformed indica cell lines compared to those of standard japonica cultivars. Two rounds of transformation were carried out for the T314 and T315 vectors.
• Transformation efficiencies using the T313, T314, and T315 vectors in BRS-CIRAD 302 were determined.
• 41, 49, and 88 primary transformants of the 3 respective populations were transferred to the containment greenhouse and grown until the harvesting of T1 seeds.
• the primary transformants are numbered after both the co-cultivated callus number (e.g., callus 21) and the hygromycin-resistant cell line number (e.g., hygromycin-resistant cell lines 21.1, 21.2 and 21.3) they originate from.
• the several hygromycin-resistant cell lines deriving from a single co-cultivated callus are generally independent transformation events 51 .
• Twenty primary transformants for each of the T314 and T315 T-DNA vectors were raised in the Kitaake cultivar using Agrobacterium-mediated transformation with the EHA 105 strain.
• T1 and T2 progeny plants for which ploidy was determined were also analyzed, along with parental and hybrid and F2 control plants, for segregation of four polymorphic micro satellites markers located in the middle of the long arms of chromosomes 1 (RM1), 8 (RM25), 9 (RM215) and 11 (RM287).
• the SSR work was carried out at the genotyping facility of Cirad in criz (France).
• DNA content of DAPi-stained cell nuclei isolated from developing leaf blades and seed endosperms was determined by FACS using a PARTEC cell analyzer and Sysmex CyStain® UV Ploidy 05-5001 buffer (www.sysmex.de) .
• the method for releasing nuclei into the buffer using manual chopping of leaf blade segments with a razor blade was that described in 9 .
• the pear-shaped developing seed was gently separated from the lemma and palea and allowed to release its milky endosperm into 0.5 ml of buffer solution using a pipette tip. Then, the turbid suspension containing nuclei was diluted in 3 ml of buffer and vortexed before FACS analysis.
• T2 plants derived from T 1 plants (n 5) of each of four TO events (T314 15.1, 37.7 and T315 5.4 and 8.1) were grown in the containment greenhouse until maturity along with fifteen BRS-CIRAD 302 Fl control plants. All the plants flowered in a synchronous manner. The following traits were recorded at the time of harvesting: Master tiller height, tiller number, antepenultimate (n-1) leaf blade length and width, flag leaf blade length and width, master tiller panicle length, number of spikelets per panicle (average of three master tillers), filled grain frequency (%), and one- thousand grain weight.
• Plant panicle fertility and pollen viability determinations The panicle fertility of Tl and T2 plants as well as that of control BRS-CIRAD 302 plants grown along the transgenic plants, was determined by averaging the filled spikelet/total spikelet ratio of the three main tillers of each plant. The panicle fertilities of four T2 progeny plants of each of five Tl plants (i.e., 20 T2 plants) were analyzed per TO event. The panicle fertility of Kitaake Tl plants of two T314 and two T315 events grown alongside wild-type plants was estimated from the master tillers of five Tl plants.
• the pollen viability was determined by counting at least 1,500 Alexander’s 59 solution-stained pollen grains released from the mature anthers of two flowers collected on four T2 plants in each of four selected TO events (T314 15.1 and 37.7, T315 5.4 and 8.1) and in control BRS-CIRAD 302. Viable pollen appear pink-colored whereas empty, unviable pollen grains appear green-colored. Alexander staining is known to overestimate pollen viability.
• Microscopy analysis of meiotic products by clearing and callose detection by aniline blue staining Ovaries and anthers collected at pre-anthesis stages (white or paleyellow anthers) from independent T2 plants of apomictic events T314 15.1 and T31437.7 (and of replicated samples collected on T3 plants of event T314 15.1) were fixed in Camoy’s or FAA fixatives and rinsed in 70% ethanol. Dissected ovaries were mounted in Hoyer’s clearing medium and gently squashed by pressure on the coverslip to expose ovules. Callose detection using aniline blue staining was performed as described 60 .
• Starch enzymatic determination Without removal of negligible soluble oligosaccharides, a dried starchy sample aliquot (25 mg) was gelatinized at 90 °C for 1.5 h with NaOH (1 mb 0.02 N) prior to being hydrolyzed at 50 °C for 1.5 h with a- amyloglucosidase in a pH 4.5 citrate buffer.
• D-glucose was then indirectly evaluated via the production of gluconate through the reduction of nicotinamide adenine dinucleotide phosphate (NADPH) using hexokinase and glucose-6-phosphate-dehydrogenase enzymes by the UV method of spectrophotometry at 340 nm (R-Biopharm, 2021).
• NADPH nicotinamide adenine dinucleotide phosphate
• R-Biopharm, 2021 The amount of starch was expressed in percent on a dry-weight basis (g starch per 100 g dried sample), using an estimated conversion factor of 0.9 between starch and D-glucose.
• Amylose calorimetric determination Amylose content was measured by differential scanning calorimetry with a DSC 8500 apparatus (Perkin Elmer, Norwalk, USA) using 9-10 mg db dried sample and 40 pL of 2% (W/v) L-a-lysophosphatidylcholine solution (Sigma Chemical Co., St Louis, USA) in a hermetically sealed micropan 62 .
• the amount of amylose was expressed in percentage, on dry starch weight basis (g amylose per 100 g dried starch).
• Table 1 Ploidy of T1 progeny plants of MiMe BRS-CIRAD 302 primary transformants harboring the sgMiMe (T313), sgMiMe_pAtECS:BBMl (T314), and sgMiMe_pOsECS:BBMl (T315) T-DNAs.
• T313 The presence of the egg cell-specific promoter:BBMl cassette was ascertained in all the T314 and T315 events.
• the number of plants analyzed varies according to T1 seed availability. Events selected for further analysis on the basis on both the score and confidence of diploid frequency appear in bold. *includes one 2n/4n chimeric plant; * includes two 2n/4n chimeric plants.
• Table 2 Frequency of diploid plants in T2 and T3 progenies of selected sgMiMe_pAtECS:BBMl (T314) and sgMiMe_pOsECS:BBMl (T315) events.
• T2 ploidy determination at least 40 progeny plants from 5 individual T1 plants (i.e., at least 200 plants per event) were analyzed.
• T3 ploidy determination 100 plants from 3 individual T2 plants (i.e., 300 plants per event) were analyzed. Observations at the T1 generation are provided to facilitate interpretation of results.
• Graham R A Proposal for IRRI to Establish a Grain Quality and Nutrition Research Center. ppl5 (2002).

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