Bioactive beads-mediated transformation of plants with large DNA fragments

Transformation of plants with large DNA fragments containing multiple genes is a promising approach to extend the possibility of plant genetic engineering. Recently we have developed a new transformation method, bioactive beads method. This method is easy, efficient and has transformation capability of large DNA fragments. However, stable transgenic plants with large DNA fragments have not been produced using this method yet.In this study, we introduced large DNA fragments (ca. 100 kb) containing Aegilops tauschii hardness genes into rice using bioactive beads method. PCR analyses showed that multiple transgenes were integrated into rice genome in all transgenic plants. The inheritance of transgenes into next generation was also confirmed. Segregation analysis showed that one of the transgenic plants was homozygous transgenic plant. The expression of transgene in this plant was detected in mRNA and protein level. This result indicated that the native promotor of the introduced gene, puroindoline b, worked in rice endosperm in tissue-specific manner. The phenotype of transgenic plants was investigated by SEM and TEM. The results showed that transgenic plants have loosely packed endosperm structure compared with that of non-transgenic plants. The analysis of milled rice flour showed that flour from transgenic rice had smaller particle size than the flour from non-transgenic plants. These results indicated that the expression of puroindoline b gave soft texture to rice grain. Our study clearly indicates that bioactive beads method is a powerful tool to introduce large DNA fragment(s) into a plant cell.