Highly efficient Agrobacterium-mediated transformation of suspension-cultured cell clusters of rice (Oryza sativa L.)

Development of a large-scale transformation procedure is essential for generating T-DNA insertion lines and FOX libraries and for gene targeting studies. Suspension culture may be one of the best sources for producing a large number of transgenic cells; however, suspension-cultured cell clusters have been reported to have low Agrobacterium-mediated transformation frequencies in rice. In this investigation, we found that rice calli produce some unidentified substance critical for Agrobacterium-mediated transformation of rice that is released in the liquid culture media. By co-cultivating 3-day old suspension-cultured cells with Agrobacterium on filter paper moistened with enriched N6 media containing suspension-cultured cell media, more than 104 stable transformants were routinely obtained from 1 g of suspension-cultured cell clusters. Transformation efficiency was about 60-times higher than that obtained from calli co-cultured in N6 media alone. Also, judging from the average weight of the cell clusters, using suspension-cultured cell clusters resulted in about 10-fold enhancement of the transformation efficiency compared with that of calli subcultured on solid media.

Research highlights▶ Our investigation has demonstrated that rice calli produced unidentified compounds that were easily diffused into medium and were critical for Agrobacterium-mediated rice transformation. The transformation efficiency of small clusters of suspension-cultured cells might have been reduced by the diffusion of these compounds in liquid medium, and the efficiency was improved by adding these compounds to the cultures. ▶ Our study represents the first use of rice suspension-cultured cell clusters co-cultivated with nurse medium resulting in high rates of Agrobacterium-mediated transformation in cell clusters of all sizes.