Session 1: Gene knockout systems using T-DNA as a mutagen

T-DNA insertional mutagenesis strategy has proven to be a powerful tool for plant functional genomics. A number of important genes have been isolated and characterized using T-DNA as a mutagen in Arabidopsis. Since highly efficient Agrobacterium-mediated transformation systems have been well established in rice, more T-DNA insertional mutagenesis projects have been initiated recently both in developed and developing countries. More than 150,000 T-DNA insertion lines have been generated so far.

In this session, three speakers talked about their progresses on T-DNA tagging and new strategies used to increase tagging efficiency.

Dr. Guiderdoni Emmanuel from CIRAD of France first presented the progresses on generating a genome-wide T-DNA insertion library. He described a highly efficient Agrobacterium-mediated transformation system with the capacity of producing 30,000 primary transformants with an average efficiency of 5 independent transformation events per co-cultured callus in one and half year. About 30% and 15% of the transformants analysed showed GUS and GFP positive, respectively. Large scale T-DNA flanking sequence rescue produced 9,700 readable sequences from 10,862 PCR products sequenced. About 90% inserts that have been mapped to chromosome 1 can be assigned to a unique location.

Dr. Su-May Yu from Institute of Molecular Biology of Academia Sinica in Taiwan reported the progresses on the new initiated project of rice functional genomics using T-DNA tagging system. Promoter-trapping and gene activation tagging systems have been established with the capacity of generating 1,000 transgenic plants per month. To handle large number of plants, bar-code system has been adopted. Meanwhile, T-DNA flanking sequences are being rescued and sequenced.

Dr. Andrew Eamens from Plant Industry CSIRO in Canberra presented a duel orientation T-DNA-cum-Ac/Ds gene trap system. With this new tagging system, firstly, the trapping and screening efficiency could be increased because of its duel orientation T-DNA gene trap housing gus in one end and gfp in the other end. A four fold increase in trapping efficiencies were observed in rice and Arabidopsis. Secondly, original T-DNA insertion could be further used as a Ds launching pad for transposon tagging. Thirdly, inclusion of a barnase killer gene in the vector backbone enabled to remove interfering VB sequences and to reduce the occurrence of complex integration patterns within screening population.