METHODS FOR PLANT TRANSFORMATION
This invention relates to methods for the transformation of Brachypodium sp. and transformed plants produced according to the method. Specifically, this invention relates to direct transformation of callus derived from immature embryos using Agrobacterium -mediated transformation, and plants regenerated from the transformed callus tissue. The methods comprise utilizing Brachypodium sp. immature embryos as the source of plant material for callus induction; induced calli can be infected by Agrobacterium hosting an appropriate binary 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 increasing the transformation frequency for transforming callus derived from immature embryos of Brachypodium sp. comprising:
(a) Producing embryogenic callus from immature embryos of Brachypodium sp.,
(b) Growing Agrobacterium cells for three days at a temperature of 19-22° C. harboring a functional plant transformation vector,
(c) Re-suspending the Agrobacterium cells in infection medium to an optical density of less than 0.25 at 600 nm,
(d) co-cultivating the resuspended Agrobacterium cells with callus tissue in infection medium containing greater than 30 g/L sucrose,
(e) culturing the infected cell on selection medium at a temperature of 25-35° C. in the dark to produce transformed tissue expressing the nucleic acid,
(f) regenerating the transformed tissue on at least one regeneration medium to produce a transformed plant,
wherein the resulting transformation efficiency is at least 15 transgenic events per immature embryo.
2 . The method of claim 1 , where the plant cells being transformed are derived from Brachypodium distachyon.
3 . The method of claim 1 , where the Agrobacterium cells are resuspended in infection medium at an optical density of 0.10-0.20.
4 . The method of claim 1 , where the Agrobacterium cells are resuspended in infection medium at an optical density of 0.13-0.17.
5 . The method of claim 1 , where the Agrobacterium cells are resuspended in infection medium at an optical density of 0.14-0.16.
6 . The method of claim 1 , where the Agrobacterium cells are resuspended in infection medium at an optical density of 0.15.