Transformation of duckweed and uses thereof
The present disclosure provides methods and compositions for genetic transformation of Lemnaceae species.
1. A method for stably transforming Lemna minor duckweed with a nucleic acid, the method comprising:
(a) inoculating, in liquid infection medium that comprises magnesium, a plant metabolizable sugar, and acetosyringone, an actively growing Lemna minor duckweed callus with an engineered Agrobacterium that comprises a nucleic acid of interest, a selectable marker gene that confers resistance to a selection substance, and a visible reporter gene encoding a protein that is visualized without killing inoculated callus, thereby producing inoculated callus;
(b) culturing inoculated callus on semi-solid nodule production medium that comprises acetosyringone, and then culturing inoculated callus on semi-solid selection medium that comprises (i) the selection substance and (ii) an antibiotic, thereby producing cultured, inoculated callus;
(c) selecting, from cultured, inoculated callus of (b), transformed callus based on expression of the visible reporter protein in the transformed callus;
(d) culturing, in liquid selection medium that comprises the selection substance and the antibiotic, selected callus of (c); and
(e) culturing callus cultured in (d) on semi-solid selection medium that comprises the selection substance and the antibiotic, thereby producing genetically engineered progeny Lemna minor duckweed comprising the nucleic acid of interest.
2. The method of claim 1 , wherein the Agrobacterium is an Agrobacterium tumefaciens.
3. The method of claim 1 , wherein the diameter of the actively growing Lemna minor duckweed callus of (a) is about 3 to 5 mm.
4. The method of claim 1 , wherein the magnesium comprises magnesium sulfate and the plant metabolizable sugar comprises sucrose.
5. The method of claim 1 , wherein the selection substance is DL-phosphinothricin.
6. The method of claim 1 , wherein the visible reporter gene encodes green fluorescent protein.
7. The method of claim 1 , wherein the semi-solid nodule production medium of (b) further comprises Murashige and Skoog basal salts, sucrose, 2,4-dichlorophenoxyacetic acid, and 6-benzylaminopurine.
8. The method of claim 1 , wherein cultured, inoculated callus of (b) is cultured for about 4 to 7 days on the semi-solid selection medium.
9. The method of claim 1 , wherein the semi-solid selection medium of (b) further comprises the selection substance, the antibiotic, basal salts and sucrose.
10. The method of claim 9 , wherein the selection substance comprises DL-phosphinothricin, and the antibiotic comprises carbenicillin and cefotaxamin.
11. The method of claim 1 , wherein the visible reporter gene encodes a fluorescent protein, and wherein (c) comprises selecting fluorescent cells using fluorescent microscopy.
12. The method of claim 1 , wherein the selected callus of (c) is cultured in liquid selection medium for about 3 to 4 weeks.
13. The method of claim 1 , wherein the liquid selection medium of (d) further comprises basal salts and sucrose.
14. The method of claim 13 , wherein the selection substance comprises DL-phosphinothricin, and the antibiotic comprises carbenicillin and cefotaxamin.
15. The method of claim 1 , wherein (e) comprises culturing callus cultured in (d) on the semi-solid selection medium until genetically engineered progeny Lemna minor duckweed is visible.
16. The method of claim 1 , wherein the semi-solid selection medium of (e) further comprises basal salts and sucrose.
17. The method of claim 16 , wherein the selection substance comprises DL-phosphinothricin, and the antibiotic comprises carbenicillin and cefotaxamin.
18. The method of claim 1 , wherein the nucleic acid of interest comprises a promoter operably linked to a nucleic acid that encodes a protein of interest or an artificial microRNA.