METHODS FOR SETARIA VIRIDIS TRANSFORMATION
This invention relates to methods for the transformation of Setaria species such as Setaria viridis and transformed plants produced according to the method. Specifically, this invention relates to direct transformation of callus derived from mature embryos using Agrobacterium -mediated transformation, and plants regenerated from the transformed callus tissue. The methods comprise utilizing Setaria mature embryos as the source of plant material for callus induction; induced calli can be infected by Agrobacterium hosting an appropriate vector. Transgenic plants are regenerated from transgenic calli grown under conditions favoring growth of transformed cells while substantially inhibiting growth of non-transformed cells. These methods provide for significantly increased plant transformation efficiency with minimal ratio of escapes.
1 . A method of transforming callus derived from mature embryos of Setaria species comprising:
(a) inducing callus growth from mature embryos at a light intensity of 5-30 μE m −2 sec −1 ,
(b) pre-treating quality callus on CSM medium for 3 to 5 days,
(c) growing Agrobacterium cells harboring a functional plant transformation vector for three days at a temperature of 19-22° C.,
(d) re-suspending the Agrobacterium cells in infection medium to an optical density of less than 1.0 at 600 nm,
(e) co-cultivating the resuspended Agrobacterium cells with callus tissue in infection medium containing greater than 30 g/L sucrose,
(f) culturing the infected cells on selection medium at a temperature of 25-35° C. for 32-49 days to produce transformed tissue expressing the nucleic acid, and
(g) regenerating the transformed tissue on at least one regeneration medium to produce a transformed plant,
wherein the resulting transformation efficiency is at least 20%.
2 . The method of claim 1 wherein said callus is derived from Setaria viridis.
3 . The method of claim 1 wherein said callus is derived from Setaria italica.
4 . The method of claim 1 wherein said inducing callus growth occurs at a light intensity of 15-25 μE m −2 sec —1 .
5 . The method of claim 1 wherein said inducing callus growth occurs at a light intensity of 10-20 μE m 2 sec 1 .
6 . The method of claim 1 wherein said Agrobacterium cells are resuspended in infection medium at an optical density of 0.10-0.24.
7 . (canceled)
8 . The method of claim 1 wherein said Agrobacterium cells are resuspended in infection medium at an optical density of 0.12-0.18.
9 . The method of claim 1 wherein said Agrobacterium cells are resuspended in infection medium at an optical density of 0.14-0.16.
10 . The method of claim 1 wherein said Agrobacterium cells are resuspended in infection medium at an optical density of 0.15.
11 . The method of claim 1 wherein said infected cells are cultured on selection medium at a temperature of 26-30° C.
12 . The method of claim 1 wherein said infected cells are cultured on selection medium at a temperature of 28° C.
13 . The method of claim 1 wherein said growing Agrobacterium cells occurs on solid YEP medium containing the appropriate antibiotics for plasmid maintenance.
14 . The method of claim 2 wherein said callus derived from Setaria viridis is derived from the accession A10.1.
15 . The method of claim 2 wherein said callus derived from Setaria viridis is derived from the accession ME034V.
16 . The method of claim 1 wherein said CSM medium does not contain a cytokinin.
17 . The method of claim 1 wherein said selection medium is CSM supplemented with appropriate chemicals to affect selection.
18 . The method of claim 1 wherein said selection medium is CIM supplemented with appropriate chemicals to affect selection.
19 . The method of claim 17 wherein said appropriate chemicals to affect selection are selected from the group of hygromycin, bialaphos, and kanamycin.
20 . The method of claim 1 wherein said co-cultivating the resuspended Agrobacterium cells occurs in the dark for three days.
21 . The method of claim 1 wherein said culturing the infected cells on selection medium occurs in the dark.