# US 11,026,381 B2 · Method for producing haploid, dihaploid and doubled haploid plants by isolated microspore culture > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,026,381 B2 - Document ID: US11026381B2 - Status: Granted - Kind: B2 - Application number: 15/747,631 - Filed: 2016-07-26 - Published: 2018-08-02 - Granted: 2021-06-08 - Earliest claimed: 2015-07-28 - Assignees: Vilmorin & Cie - Applicants: Vilmorin & Cie - Inventors: Jianxin Chen; Elise Vanek; Mark Pieper - Web page: https://patentgrep.com/patent/US11026381B2 ## CPC - A01H 4/005: {Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques} - A01H 3/04: by treatment with chemicals - A01H 4/008: {Methods for regeneration to complete plants} - A01H 6/34: Cucurbitaceae, e.g. bitter melon, cucumber or watermelon - A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES ## Abstract The present invention relates to a method for producing haploid, dihaploid, polyhaploid and/or doubled haploid plants of the family Cucurbitaceae from isolated microspores, wherein said method comprises a) culturing isolated microspores to obtain embryos competent for plant regeneration, wherein the microspores have been isolated from plant material of a donor plant of the family Cucurbitaceae; and b) regenerating plants from the embryos; wherein step (a) comprises contacting the microspores with one or more inhibitor of histone deacetylase (HDACi) and one or more polyamine. The present invention also relates to a method for producing haploid, dihaploid, polyhaploid and/or doubled haploid embryos, to related kits and compositions, and to plants obtained according to the methods. ## Claims ### Claim 1 A method for producing haploid, dihaploid, polyhaploid and/or doubled haploid plants of the family Cucurbitaceae from isolated microspores, wherein said method comprises: a) culturing isolated microspores to obtain embryos competent for plant regeneration, wherein the microspores have been isolated from plant material of a donor plant of the family Cucurbitaceae; and b) regenerating plants from the embryos; wherein step (a) comprises contacting the microspores with one or more inhibitor of histone deacetylase (HDACi) and one or more aliphatic polyamine. ### Claim 2 (depends on 1) The method of claim 1, wherein step (a) comprises inducing the sporophytic development of the microspores in a culture medium comprising the HDACi and subsequently adding the polyamine in the culture of isolated microspores. ### Claim 3 (depends on 2) The method of claim 2, wherein the polyamine is added no sooner than the isolated microspores have divided in culture. ### Claim 4 (depends on 1) The method of claim 1, wherein the HDACi is selected from hydroxamic acids, cyclic tetrapeptides, depsipeptides, aliphatic acids, benzamides, electrophilic ketones, and mixtures thereof. ### Claim 5 (depends on 4) The method of claim 4, wherein the HDACi is selected from suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA) and mixtures thereof. ### Claim 6 (depends on 1) The method of claim 1, wherein the polyamine is selected from putrescine, spermidine, spermine, and mixtures thereof. ### Claim 7 (depends on 1) The method of claim 1, wherein said step (a) comprises: (i) culturing the isolated microspores to obtain globular embryos; and (ii) culturing the globular embryos to obtain elongated embryos; wherein step (ii) comprises at least one of: culturing the globular embryos in the presence of an adsorbent material; culturing the globular embryos in a multilayer culture system, comprising a liquid phase overlaying a solid phase; culturing the globular embryos under agitation; and culturing the globular embryos in the dark. ### Claim 8 (depends on 7) The method of claim 7 wherein the solid phase comprises activated charcoal and/or the liquid phase comprises at least one of abscisic acid and one or more polyamine. ### Claim 9 (depends on 1) The method of claim 1, wherein the plant material of the donor plant has been obtained through the steps of: growing a donor plants of the family Cucurbitaceae; subjecting the donor plant to a stress treatment; and recovering plant material containing microspores at a developmental stage competent for induction of embryo development. ### Claim 10 (depends on 1) The method of claim 1, wherein the microspores have been isolated from the plant material of the donor plant at a mid-uninucleate to early binucleate stage of development. ### Claim 11 (depends on 1) The method of claim 1, wherein the isolated microspores have been subjected to a stress treatment, prior to step (a). ### Claim 12 (depends on 1) The method of claim 1, wherein the donor plant belongs to the genera Cucumis, Cucurbita or Citrullus. ### Claim 13 (depends on 1) The method of claim 1, wherein the donor plant is parthenocarpic, and/or wherein the donor plant is selected from a monoecious, dioecious, gynoecious, androecious, andromonoecious, gynomonoecious, hermaphrodite, protoandrous or protogynous plant. ### Claim 14 (depends on 1) The method of claim 1, further comprising a step of doubling of the chromosome stocks of the haploid plants regenerated at step (b). ### Claim 15 A method for producing haploid, dihaploid, polyploid and/or doubled haploid embryos of the family Cucurbitaceae from isolated microspores, wherein said method comprises: a) culturing isolated microspores to obtain embryos, wherein the microspores have been isolated from plant material of a donor plant of the family Cucurbitaceae, wherein step (a) comprises contacting the microspores with one or more inhibitor of histone deacetylase (HDACi) and one or more aliphatic polyamine. ### Claim 16 (depends on 1) The method of claim 1, further comprising the step of crossing a plant regenerated at step (b), with another plant of the family Cucurbitaceae and obtaining seeds and/or progeny plants.