The angle between the second lamina and sheath was measured. An F2 mapping population from a cross between gsor300084 and the indica variety Dular was generated. InDel markers (170 pairs) and 20 F2 individuals showing mutant phenotypes were used in primary mapping. Seven InDel markers were developed and 358 F2 individuals were used for fine mapping. Genomic DNA fragments of the D61 gene from Matsumae and gsor300084 were amplified and sequenced. The coding sequence of the D61 gene from the wild type and the gsor300084 mutant
was fused in-frame to the 3′ end of the sGFP gene in the transient expression vector pCAMBIA1205-GFP. The 1205-GFP-d61300084 and 1205-GFP-D61 fusion constructs were transformed into protoplasts prepared from wild-type rice seedlings by polyethylene glycol treatment. The transformed protoplasts were Selleck INCB018424 incubated at 28 °C for 16 h. Green fluorescence of the GFP fusion protein was observed under a Zeiss LSM 510 META confocal microscope. The phenotype of the gsor300084 mutant was different from that of the wild type variety Matsumae in many respects. PLX3397 supplier The plant height of gsor300084 was less than that of Matsumae from the seedling stage ( Fig. 1-A). At maturity, the plant height of gsor300084
was only about one half that of the wild type ( Fig. 1-B and Table 1). In wild-type plants, the leaf blade bent away from the vertical axis of the leaf sheath toward the abaxial side, but in gsor300084 most of the leaves were erect ( Fig. 1-D). The panicle of gsor300084 was smaller than that of the wild type ( Fig. 1-E). Moreover, the grains of gsor300084 were smaller and rounder ( Fig. 1-F and Table 1) and the grain weight was significantly reduced ( Table 1). Internode triclocarban elongation was severely inhibited ( Fig. 1-G) except for the uppermost internode ( Fig. 1-H), indicating that gsor300084 is a d6-type dwarf mutant
[28]. In rice mutants with defects in BR biosynthesis or sensitivity, elongation of the mesocotyl is inhibited when plants are grown in complete darkness [2]. To determine whether gsor300084 is a BR-related mutant, the mesocotyl internode elongation pattern in the darkness was observed. After growth in complete darkness for two weeks, the wild type plants exhibited mesocotyl elongation, whereas no such elongation occurred in gsor300084 ( Fig. 2). The failure of mesocotyl elongation in gsor300084 is similar to the phenotype of other rice BR-related mutants grown in darkness [4] and [29]. Based on the abnormal phenotypes of gsor300084, we suspected that the gsor300084 mutant was defective in BR biosynthesis or sensitivity. To identify the type of BR mutant to which gsor300084 belongs, we evaluated the coleoptile elongation and root length of wild type and 300084 seedlings in response to BL. Rice seeds were germinated in half-strength MS medium with 0 or 1 μmol L− 1 BL in complete darkness.