Establishment of a plant tissue culture system and genetic transformation for agronomic improvement of Indonesian black rice (Oryza sativa L.)

Abstract

Indonesian black rice has the potential to be developed as a functional food due to their high anthocyanin content and other nutritional benefits such as enhanced Fe and amino acid composition. Black rice productivity, however, is low due to its long harvesting time, caused by late heading date, and therefore poor yield. These negative traits in black rice may be improved using conventional and modern approaches by manipulating genes of interest. As a tool to study functional genomics in black rice, genetic transformation was studied. A suitable in vitro regeneration method, previously unavailable for Indonesian black rice cultivars, first had to be established as a key prerequisite for genetic transformation. The purpose of this study therefore was to establish an efficient method for culturing Indonesian black rice cultivars and subsequently to use the Hd3a flowering gene under the RPP16 promoter as a relevant gene to improve the flowering period with either in vitro or in planta transformation methods. We found that the in vitro regeneration method established was able to generate fertile black rice plants and furthermore enabled the generation of transgenic plants. T0 transgenics harboring the Hd3a gene driven by the RPP16 promoter yielded extremely early flowering, dwarfed, and low-yielding plants, but we obtained a normal phenotype with early flowering in the T1 generation. In vitro transformation was more efficient in terms of time taken and number of positive transformants than in planta transformation for genetic transformation of Indonesian black rice by Agrobacterium-mediated transformation. Indonesian black rice requires a specific temperature range of 30–32 oC during the tissue culture phase for successful genetic transformation.