D Fe accumulated well in a restricted number of seeds. The seeds of your Fer-NAS-NAAT-IDS3 lines accumulated both Fe and Zn. In contrast, Fer line 13-6 did not contain higher levels of Zn in its seeds, as when compared with the NT line (Figure 9). Masuda et al. (2008) showed that the insertion of IDS3 in to the rice genome enhanced the Zn concentration by 35 in polished seeds and by 29 in brown seeds in an Andosol field experiment. Suzuki et al. (2008) also showed that the Zn concentration was increased by 37 in brown seeds inside a calcareous soil field experiment. Hence, the enhanced Zn concentration in the FerNAS-NAAT-IDS3 lines might have been brought on by the introduction of IDS3.Classic breeding has also created high Fe rice which include IR68144. Hence, for further improvement of Fe concentration in seeds, it will be additional effective to generate greater Fe biofortified rice by transgenic technique employing a higher Fe selection which has currently been created by regular breeding. In conclusion, transgenic rice expressing each ferritin plus the barley MA synthase gene IDS3 showed elevated Fe concentration when the plants were cultivated in each commercially supplied soil and calcareous soil. Fe-deficiency tolerance was also noted below calcareous soil cultivation. These outcomes indicate that the concomitant introduction of ferritin and IDS3 is definitely an successful method to biofortify seeds with Fe with out causing Fe-deficiency symptoms below Fe-limited circumstances. This system will likely be specifically advantageous for use in Fe-limited environments, like these using a higher soil pH and upland cultivation.ACKNOWLEDGMENTSWe thank Dr. Mitsuru Abo (The University of Tokyo) for technical help for ICP-AES analysis, Dr. Kyoko Higuchi and Dr. Teppei Maruyama (Tokyo University of Agriculture) for help in rice cultivation. We also thank Ms. Reiko Nakanishi Itai and Dr. Motofumi Suzuki (The University of Tokyo) for technical tips concerning Northern blot analysis. We thank Dr. Fumiyuki Goto (CRIEPI, Japan) for providing soybean ferritin antibody, and Dr. Toshihiro Yoshihara (CRIEPI, Japan) and Dr. Fumio Takaiwa (NIAS, Japan) for giving OsGlb1 promoter, OsGluB1 promoter, and SoyferH2 gene. This work was supported by the Harvest Plus project, Core Analysis for Evolutional Science and Technologies (CREST), plus the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.Price of 2,6-Bis(aminomethyl)pyridine SUPPLEMENTARY MATERIALThe Supplementary Material for this short article could be identified on the web at: http://frontiersin.856562-91-9 uses org/Plant_Physiology/10.3389/ fpls.2013.00132/abstractHiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Effective transformation of rice (Oryza sativa L.PMID:23715856 ) mediated by Agrobacterium and sequence evaluation with the boundaries in the T-DNA. Plant J. 6, 271?82. Higuchi, K., Suzuki, K., Nakanishi, H., Yamaguchi, H., Nishizawa, N. K., and Mori, S. (1999). Cloning of nicotianamine synthase genes, novel genes involved within the biosynthesis of phytosiderophores. Plant Physiol. 119, 471?79. Higuchi, K., Watanabe, S., Takahashi, M., Kawasaki, S., Nakanishi, H., Nishizawa, N. K., et al. (2001). Nicotianamine synthase gene expression differs in barley and rice below Fe-deficient situations. Plant J. 25, 159?67. Inoue, H., Higuchi, K., Takahashi, M., Nakanishi, H., Mori, S., and Nishizawa, N. K. (2003). Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in longdistance transport of iron and differentially regulated by iron. Plant J. 36.