The polyembryo gene (OsPE) in rice.

T-DNA insertional mutagenesis is one of the most important approaches for gene discovery and cloning. A fertile polyembryo mutant generated by T-DNA/Ds insertion in Oryza sativa, cv. Basmati 370 showed twin or triple seedlings at a frequency of 15-20%. T-DNA insertion was confirmed by 950 bp hpt gene amplification in the promoter region of the candidate gene. The annotated protein corresponding to the OsPE candidate gene has been reported as a hypothetical protein in O. sativa. OsPE gene lacked functional homologs in other species. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. RT-PCR showed the expression of OsPE gene in Basmati 370 shoots. Full-length OsPE gene was cloned in Basmati 370. The combined use of Southern blot, genome walking, TAIL-PCR, RT-PCR techniques, and bioinformatics led to the identification of a candidate gene controlling the multiple embryos in rice. There is gain of function, i.e., multiple embryos in the seeds in the knockout mutant OsPE whereas its wild-type allele strictly controls single embryo per seed. The seeds with multiple embryos are distributed at random in the rice mutant panicle. The origin of multiple embryos, whether apomictic, zygotic or both is under investigation.

A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion.

A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using Hm(R) Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA(3)) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F(2) from OsAPC6 x Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.