About Insertion Mutagenesis in Rice

International mutant libraries
In the last decade the use of insertion mutant libraries has largely contributed to the discovery and validation of gene function, notably in Arabidopsis and maize. Insertional mutagenesis is -together with chemical/physical mutagenesis, RNA interference (Waterhouse and Helliwell 2003 Nature Review Genetics, 4: 29-38) and the versatile but still lowly efficient process in higher plants, homologous recombination (Hanin and Paszkowski 2003 Curr Opin Plant Biol., 67: 157-162) - one of the methods to inactivate gene expression. It relies on the random insertion of a foreign DNA in the genome which systematically disrupts any gene and further acts as a molecular tag. The insertional mutagen can be the T-DNA transferred and randomly integrated during co-culture of plant cells with Agrobacterium or an endogenous or introduced transposable element (transposon or retrotransposon).
Large collections of several hundred thousands T-DNA, Ac/Ds and En/Spm insertion lines are now available in Arabidopsis - In combination, they likely contain enough inserts to find an insertion in any gene -. These lines are used for direct genetics screens under normal or specific growth conditions, as well as for reverse genetics screens allowing PCR-based or in silico isolation in DNA pools and FST (a sequence of the genome flanking the mutagen insertion site) databases respectively, for individual plants that carry a particular mutation in a gene of interest.

In the last 6 years, aside from efforts to produce conventional populations using chemical or physical mutagens ( Wu et al. 2005 Plant Mol Biol, 59:85-97 ), several initiatives to develop gene machines through insertional mutagenesis have been launched in rice ( Hirochika et al. 2004 Plant Mol Biol, 54:325-334). These use the Maize Ac element (Enoki et al., 1999 Plant J. 19:605-613, Chin et al., 1999 Plant J: 19 615-623, Greco et al 2003 Theor Appl Genet. 108:10-24.; Upadhyaya et al 2002 Funct Plant Biol 29:547-559; Kolesnik et al 2004 Plant J, 37:301-314; Guo et al. 2003 Theor Appl Genet.; Van enckevort et al. 2005 Plant Mol Biol 2005 Sep;59(1):99-110.), the maize En/Spm element (Kumar et al. 2005 Plant J 44:879-892) ,the tissue culture-activated endogenous retrotransposon Tos 17 ( Miyao et al. 2003 Plant Cell 15:1771-1780 ) or T-DNAs carrying a gene trap (Jeon et al 2000, Plant J, 22:561-570; Sallaud et al. 2004 Plant J, 39:450-464 ) or an activation tagging system (Jeong et al., 2002 Plant Physiol, 130:1636-1644; Jeong et al. 2006 Plant J, 45:123-132; Hsing et al. 2006 Plant Mol Biol. ). The most advanced initiatives are those of NIAS Tsukuba, Japan and POSTECH Pohang, Korea. 50,000 lines totalizing 250,000 Tos17 insertions have been produced at NIAS. A database of 18,000 Tos17 flanking sequences is available for similarity search through BLAST queries at Tos17 website. At POSTECH more than 100,000 T-DNA insertion lines and 80,000 FST have been produced at POSTECH RISD . These initiatives are now merging their efforts in an International Rice Functional Genomics Consortium, coordinated by IRRI. Current 2006 status of the production of insertion lines and contacts is provided in table 1.