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US-20260125696-A1 - EFFICIENT INDUCTION OF PARTHENOGENESIS IN CROP PLANTS

US20260125696A1US 20260125696 A1US20260125696 A1US 20260125696A1US-20260125696-A1

Abstract

Methods for improving parthenogenesis efficiency by DWT1 and BABY BOOM transcription factors in plants are provided. A rice embryo trigger transcription factor BABY BOOM1 can initiate embryogenesis when expressed in the unfertilized egg cell through a process called parthenogenesis (Khanday et al., 2019. Nature 565: 91-95). The parthenogenesis efficiency by BABY BBOM1 itself is 10-29%. This invention describes methods of high frequency of parthenogenesis by simultaneous expression of BABY BOOM and DWT1 transcription factors. When BABY BOOM1 and DWT1 are expressed together through egg cell-specific promoters, parthenogenesis efficiencies of up to 90% are achieved. These high parthenogenesis efficiencies are a prerequisite for field applications of synthetic apomixis in crop plants.

Inventors

  • Imtiyaz KHANDAY
  • Hui Ren
  • Venkatesan Sundaresan

Assignees

  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260507
Application Date
20230929

Claims (19)

  1. 1 . A plant comprising: a first expression cassette comprising a first plant egg-specific promoter operably linked to a polynucleotide encoding a Dwarf Tiller 1 (DWT1) polypeptide; and a second expression cassette comprising a second plant egg-specific promoter operably linked to polynucleotide encoding a Babyboom polypeptide, wherein the plant has more efficient parthenogenesis than a control plant lacking at least the first, and optionally the second, expression cassette.
  2. 2 . The plant of claim 1 , wherein the plant is diploid and progeny from the plant resulting from parthenogenesis are haploid.
  3. 3 . The plant of claim 1 , wherein the plant further comprises sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype such that the plant produces clonal seed.
  4. 4 . The plant of claim 3 , wherein the MiMe expression cassettes comprise: an expression cassette comprising a promoter operably linked to a gRNA that targets OSD1 or an ortholog thereof; an expression cassette comprising a promoter operably linked to a gRNA that targets ATREC8 or an ortholog thereof; an expression cassette comprising a promoter operably linked to a gRNA that targets SPO11, or PRD1, or PRD2 or PRD3/PAIR1 or an ortholog thereof.
  5. 5 . The plant of claim 1 , wherein the DWT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:33.
  6. 6 . The plant of claim 1 , wherein the DWT1 polypeptide comprises an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6 or 72.
  7. 7 . The plant of claim 1 , wherein the Babyboom polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 10-29.
  8. 8 . The plant of claim 1 , wherein the first egg-specific promoter and the second egg-specific promoter are the same.
  9. 9 . The plant of claim 1 , wherein the first egg-specific promoter and the second egg-specific promoter are the different.
  10. 10 . The plant of claim 1 , wherein the first egg-specific promoter and the second egg-specific promoter or both comprise SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.
  11. 11 . The plant of claim 1 , wherein the plant is a rice plant.
  12. 12 . A method of making the plant of claim 1 , the method comprising, introducing the first expression cassette and the second expression cassette into the plant.
  13. 13 . The method of claim 12 , wherein the introducing comprises transformation of the plant with the first or second or both expression cassettes, introducing the first or second or both expression cassettes into the plant with a sexual cross, or introducing one of the first and second expression cassettes into the plant via transformation and introducing one of the first and second expression cassettes into the plant via a sexual cross.
  14. 14 . A method of generating haploid progeny, the method comprising cultivating a plant of claim 1 ; and collecting haploid seed from the plant.
  15. 15 . A method of generating clonal progeny, the method comprising growing a plant of claim 1 , and collecting clonal seed from the plant.
  16. 16 . A nucleic acid comprising an expression cassette comprising a plant egg-specific promoter operably linked to a polynucleotide encoding a DWT1 polypeptide.
  17. 17 . The nucleic acid of claim 16 , wherein the promoter comprises SEQ ID NO: 30, SEQ ID NO:31 or SEQ ID NO:32.
  18. 18 . The nucleic acid of claim 16 , wherein the DWT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:33.
  19. 19 . The nucleic acid of claim 16 , wherein the DWT1 polypeptide comprises an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6 or 72.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS The present patent application is a US National Phase Application Under 371 of International Application PCT/US2023/034142 filed Sep. 29, 2023, which claims benefit of priority to U.S. Provisional Patent Application No. 63/412,666, filed Oct. 3, 2022, which are incorporated by reference for all purposes. REFERENCE TO A SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 21, 2023, is named 081906-1409451-251010PC_SL.xml and is 147,326 bytes in size. BACKGROUND OF THE INVENTION Fusion of haploid gametes—an egg and a sperm during fertilization initiates embryogenesis in sexually reproducing plants. The molecular basis of embryo initiation after fertilization is still obscure. Previous transcriptome analysis of rice gametes and zygotes identified two transcription factors OsBBM1 and OsDWT1, belonging to the PLETHORA/BABY BOOM clade of APETALA2-family and WUSCHEL-Homebox or WOX family, respectively that are only expressed from the male alleles in the zygote after fertilization (Anderson et al., Developmental Cell 2017, 43:349-358.e4 (2017)). It was further shown that OsBBM1 functions to initiate embryogenesis after fertilization in rice plants. In transgenic rice with OsBBM1 expressed under an egg cell-specific promoter, the result is parthenogenesis (embryo development without fertilization) and the production of haploid progeny (Khanday et al., Nature 565:91-95 (2019)). The parthenogenesis frequencies arising from ectopic expression of OsBBM1 in the egg cell were found to be in the range of 10-29% (Khanday et al., Nature 565:91-95 (2019)). BRIEF SUMMARY OF THE INVENTION In some embodiments, a plant is provided comprising: a first expression cassette comprising a first plant egg-specific promoter operably linked to a polynucleotide encoding a Dwarf Tiller 1 (DWT1) polypeptide; and a second expression cassette comprising a second plant egg-specific promoter operably linked to polynucleotide encoding a Baby boom polypeptide, wherein the plant has more efficient parthenogenesis than a control plant lacking at least the first, and optionally the second, expression cassette. In some embodiments, the plant is diploid and progeny from the plant resulting from parthenogenesis are haploid. In some embodiments, the plant further comprises sufficient mitosis instead of meiosis (MiME) expression cassettes comprising a promoter operably linked to gRNAs to induce a MiME phenotype such that the plant produces clonal seed. In some embodiments, the MiMe expression cassettes comprise: an expression cassette comprising a promoter operably linked to a gRNA that targets OSD1 or an ortholog thereof: an expression cassette comprising a promoter operably linked to a gRNA that targets ATREC8 or an ortholog thereof: an expression cassette comprising a promoter operably linked to a gRNA that targets SPO11, or PRD1, or PRD2 or PRD3/PAIR1 or an ortholog thereof. In some embodiments, the DWT1 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:33. In some embodiments, the DWT1 polypeptide comprises an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6 or 72. In some embodiments, the Baby boom polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 98% identical to SEQ ID NO:10-29. In some embodiments, the first egg-specific promoter and the second egg-specific promoter are the same. In some embodiments, the first egg-specific promoter and the second egg-specific promoter are the different. In some embodiments, the first egg-specific promoter and the second egg-specific promoter or both comprise SEQ ID NO:30, SEQ ID NO: 31 or SEQ ID NO:32. In some embodiments, the plant is a rice plant. Also provided is a method of making the plant as described above or elsewhere herein. In some embodiments, the method comprises, introducing the first expression cassette and the second expression cassette into the plant. In some embodiments, the method further comprises selecting plant cells, tissues, or plants with the introduced expression cassettes, and optionally regenerating plants from the selected plant cells, tissues, or plants. In some embodiments, the introducing comprises transformation of the plant with the first or second or both expression cassettes, introducing the first or second or both expression cassettes into the plant with a sexual cross, or introducing one of the first and second expression cassettes into the plant via transformation and introducing one of the first and second expression cassettes into the plant via a sexual cross. Also provided is a method of generating haploid progeny (or progeny having half the ploidy of the parent plant(s)). In some embodiments, the method comprises cultivating a pl