RESEARCH ARTICLE Studies of mitochondrial morphology and DNA amount in the rice egg cell Hideki Takanashi • Takayuki Ohnishi • Mirai Mogi • Takashi Okamoto • Shin-ichi Arimura • Nobuhiro Tsutsumi Received: 15 July 2009 / Revised: 15 October 2009 / Accepted: 7 November 2009 / Published online: 5 December 2009 Ó Springer-Verlag 2009 Abstract In plant vegetative cells, mitochondria are usually small and grain-shaped. In contrast, unusually shaped giant mitochondria (large cup-shaped or long stretched-rod-shaped) appear in the egg cells of geranium, maize, Ipomoea nil, and bracken. In this study, to charac- terize egg cell mitochondria in rice, we used nonenzymatic manual dissection to isolate unfertilized egg cells of rice and observed the egg cell mitochondria and mitochondrial DNA (mtDNA) simultaneously. These observations showed that the mitochondria in the rice egg cell are small and grain-shaped, unlike the mitochondria in geranium, maize, I. nil, and bracken. Double staining of mitochondria by MitoTracker and mtDNA by SYBR Green I showed that mitochondria in the rice egg cell have a large amount of mtDNA compared with the rice root protoplast. We also used real-time PCR analysis to quantify the mtDNA amount in the rice egg cell. We quantified the copy num- bers of four mitochondrial genes per single rice egg cell and rice leaf protoplast. Real-time PCR analysis revealed that the egg cell has more than ten times more copy numbers of all of four genes encoded in the mitochondrial genome compared with the leaf protoplast. Keywords Rice Á Egg cell Á Mitochondria Á Mitochondrial DNA Introduction In the mature embryo sac of many higher plants, repro- ductive cells, such as the egg cell, central cell, and syn- ergids in the micropylar area, have very different cell structures (Jensen 1965; Schulz and Jensen 1968). This difference is also observed in intracellular organs such as mitochondria. Plant mitochondria usually appear as small organelles; however, large networked mitochondria appear in the shoot apical meristem of Arabidopsis thaliana (Segui-Simarro et al. 2008) and Citrullus vulgaris (Bendich and Gauriloff 1984). Furthermore, the unusually shaped giant mitochondria, which have a large cup shape or a long, stretched-rod shape, appear in the egg cells of Pelargonium zonale (Kuroiwa and Kuroiwa 1992; Kuroiwa et al. 1996), Zea mays (Diboll and Larson 1966; Faure et al. 1992; Mo ´l et al. 2000), Pharbitis purpurea (Hu and Hu 1996), and Pteridium aquilinum (Tourte 1975). These giant mito- chondria have been studied thoroughly in P. zonale. Using a4 0 ,6-diamidino-2-phenylindole (DAPI)-staining method, Kuroiwa et al. (1996) reported that a stack of cup-shaped giant mitochondria in the mature egg cell contain more than 300 Mbp of DNA, whereas one mitochondrion in the embryo sac mother cell contains only 0.34 Mbp of DNA. Communicated by R. Bock. Electronic supplementary material The online version of this article (doi:10.1007/s00294-009-0277-3) contains supplementary material, which is available to authorized users. H. Takanashi Á T. Ohnishi Á M. Mogi Á S. Arimura Á N. Tsutsumi (&) Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan e-mail: atsutsu@mail.ecc.u-tokyo.ac.jp H. Takanashi e-mail: aa077003@mail.ecc.u-tokyo.ac.jp T. Okamoto Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-osawa, Hachioji, 192-0397 Tokyo, Japan 123 Curr Genet (2010) 56:33–41 DOI 10.1007/s00294-009-0277-3