Graft-mediated hybridisation of monocotyledonous plants

Patent metadata

Field	Value
Publication number	US-2022015311-A1
Application number	US-201917293765-A
Country	US
Kind code	A1
Filing date	Nov 14, 2019
Priority date	Nov 14, 2018
Publication date	Jan 20, 2022
Grant date	—

Abstract

The invention relates to the production of graft-mediated hybrid monocotyledonous plants. Methods for the production of such plants are disclosed herein.

First claim

1 . A method of producing a hybrid monocot tissue or plant, comprising:
(a) providing a first tissue comprising:
(i) mesocotyl and radicle tissue; or
(ii) mesocotyl and plumule tissue
of a first monocot plant, wherein said first tissue comprises in its genome a first marker; (c) providing a second tissue comprising:
(i) mesocotyl and radicle tissue; or
(ii) mesocotyl and plumule tissue
of a second, different monocot plant, wherein said second tissue comprises in its genome a second, different marker; (c) placing said first tissue in contact with said second tissue; (d) allowing fusion of the first and second tissues such that a graft junction forms, wherein said graft junction comprises at least one hybrid cell comprising said first and second markers; (e) selecting said at least one hybrid cell based on the presence of said first and second markers; and (f) regenerating a hybrid monocot tissue or plant from said at least one hybrid cell.

2 . The method of claim 1 , wherein said first tissue comprises mesocotyl and plumule tissue and said second tissue comprises mesocotyl and radicle tissue.

3 . The method of claim 1 or 2 , wherein after step (d) and before step (e), said graft junction is cut to provide at least one longitudinal graft junction section, wherein said graft junction section comprises said at least one hybrid cell comprising said first and second markers.

4 . The method of any one of claims 1 to 3 , wherein said first marker and/or said second marker is a selectable marker, wherein said selectable marker is selectable with a selection agent.

5 . The method of any one of claims 1 to 4 , wherein said first marker and/or said second marker is a visual marker; preferably wherein said visual marker is:
(i) a fluorescent marker;
(ii) GUS; or
(iii) luciferase

6 . The method of any one of claims 1 to 5 , wherein said first marker and/or said second marker is an endogenous trait, wherein said endogenous trait is not endogenous to both the first tissue and the second tissue; preferably wherein said endogenous trait confers:
(i) salinity tolerance;
(ii) chilling tolerance;
(iii) freezing tolerance;
(iv) heavy metal detoxification;
(v) explosive detoxification; or
(vi) the ability to metabolise mannose, xylose or benzyladenine-N-3-glucuronide.

7 . The method of any one of claims 1 to 4 , wherein said first marker and said second marker are selectable markers, wherein said first selectable marker is selectable with a first selection agent and said second selectable marker is selectable with a second, different selection agent.

8 . The method of claim 4 or 7 , wherein said first selection agent and/or said second selection agent comprises:
(i) an antibiotic;
(ii) a herbicide;
(iii) a toxin;
(iv) salt; or
(v) a sugar.

9 . The method of claim 8 , wherein:
(i) said antibiotic is selected from the group consisting of: kanamycin, geneticin (G418), hygromycin, paramomycin, neomycin, spectinomycin, streptomycin, gentamicin, tobramycin, apramycin, bleomycin, phleomycin, streptothricin, and/or chloramphenicol; (ii) said herbicide is selected from the group consisting of: glyphosate, glufosinate (or phosphinothricin/BASTA), bialaphos, chlorsulfuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), atrazine, paraquot, methyl viologen, metsulfuron-methyl, triclopyr, 3-Amino-1,2,4-triazole (3-AT), a sulfonylurea, an imidazolinone, a triazolopyrimidine, a pyrimidinyl oxybenzoate, and/or a sulfonylamino carbonyl triazolinone; (iii) said toxin is selected from the group consisting of: 2-deoxyglucose, thialysine, methotrexate, cyanamide, sodium hypochloride, gabaculine, 4-methyl tryptophan, and/or glycine betaine aldehyde; and/or (iv) said sugar is selected from the group consisting of: mannose, xylose and benzyladenine-N-3-glucuronide.

10 . The method of any one of claims 7 to 9 , wherein said regenerating comprises:
(i) placing said graft junction or graft junction section on a base medium comprising at least one auxin, at least one cytokinin and said first and second selection agents; preferably for one month to one year; such that hybrid tissue forms from said hybrid cell; and optionally
(ii) placing said hybrid tissue on a shooting medium; preferably for one to two months; such that at least one hybrid shoot forms from said hybrid tissue; and further optionally
(iii) placing said hybrid shoot on rooting medium; preferably for two to four weeks; such that at least one hybrid monocot plant comprising roots forms; and further optionally
(iv) transferring said hybrid monocot plant to soil.

11 . The method of any one of claims 1 to 10 , wherein the first tissue and/or the second tissue are provided:
(i) from a first seed and a second seed; and/or
(ii) by micropropagation in tissue culture.

12 . The method of claim 11 , wherein said first seed and/or said second seed comprises its seed coat.

13 . The method of claim 11 or 12 , wherein said first seed and/or said second seed:
(i) is dry or imbibed in water; preferably imbibed in water for 8 to 72 hours; and/or
(ii) is partially germinated in the dark; preferably for 4 to 30 days.

14 . The method of claim 11 or 12 , wherein said first seed is imbibed in water; preferably for 8to 72 hours; and said second seed is dry.

15 . The method of any one of claims 11 to 14 , wherein:
(i) said first and/or said second seed is transversely cut; preferably with a razor or other straight blade; more preferably wherein the razor blade is 0.1 to 0.2 mm thick; and/or
(ii) said first and/or said second seed is cut with a tissue puncher; preferably wherein the tissue puncher is 1.2 mm in diameter and comprises a cutting edge with a thickness of 0.1 to 0.3 mm.

16 . The method of any one of claims 11 to 15 , wherein the first tissue and/or the second tissue is tissue cultured on nutrient medium containing a cytokinin; preferably wherein the cytokinin is:
(i) N 6 -benzyladenine (BAP);
(ii) kinetin;
(iii) zeatin;
(iv) dihydrozeatin;
(v) thidiazuron (TDZ);
(vi) diphenylurea;
(vii) 6-benzylamino-9-(2-tetrahydropyranyl)-9H-purine (BPA);
(viii) forchlorfenuron; or
(ix) N 6 -isopentenyladenine (2iP).

17 . The method of any one of claims 1 to 16 , wherein the first tissue and the second tissue are from the same species.

18 . The method of any one of claims 1 to 16 , wherein the first tissue and the second tissue are from different species within the same order.

19 . The method of claim 18 , wherein either one of the first tissue and the second tissue is from a C 3 plant and the other one is from a C 4 plant; preferably wherein the C 3 plant is selected from the group consisting of: bread wheat ( Triticum aestivum ) and rice ( Oryza sativa ) and/or the C 4 plant is selected from the group consisting of: pearl millet ( Pennisetum glaucum ), sorghum ( Sorghum bicolor ) and maize ( Zea mays ); more preferably wherein the trait of C 4 photosynthesis is conferred to the regenerated hybrid monocot tissue or plant.

20 . The method of claim 18 , wherein either one of the first tissue and the second tissue is:
(i) from a salinity tolerant plant and the other is from a salinity intolerant plant; preferably wherein the salinity tolerant plant is selected from the group consisting of: Oryza coarctata, sea barley grass ( Hordeum marinum ) and tall wheatgrass ( Thinopyrum ponticum or Agropyron elongatum ) and/or the salinity intolerant plant is selected from the group consisting of: rice (

Assignees

• Cambridge Entpr Ltd

Inventors

• Reeves Gregory
• Hibberd Julian

Classifications

• A01H1/06 Primary

  Processes for producing mutations, e.g. treatment with chemicals or with radiation (specific mutations prepared by genetic engineering on plant cell or plant tissues C12N15/00 {; process for producing transgenic plants C12N15/82}) · CPC title

• A01H6/4678

  Triticum sp. [wheat] · CPC title

• A01H6/4636

  Oryza sp. [rice] · CPC title

• A01H6/4654

  Pennisetum [pearl millet] · CPC title

• A01H6/4624

  Hordeum vulgarus [barley] · CPC title