ABSTRACT
T-DNA mediated gene tagging, a technique pioneered in Arabidopsis thaliana, has become an increasingly valuable approach for gene identification and isolation. Several tissue culture and whole plant transformation protocols have been developed to introduce the T-DNA into the plant genome. More than 20000 independent transformants have been generated with an average of 1.5 inserts each. Screening of these lines for visible alterations in phenotype has resulted in approximately 4000 mutants. Forty percent of these mutations co-segregate with the selectable marker in the T-DNA. Several dozen genes have been cloned and characterized from mutants in various biochemical and developmental pathways. The T-DNAs have been found to insert into the gene and the genome of Arabidopsis in a random manner. This large population of transformants is now being utilized in a reverse genetics approach to identify insertion mutants for sequences of interest. Primers from the T-DNA borders and the gene of interest are used in PCR reactions against DNA extracted from pools of 100 transformants in each of two dimensions. A PCR product in each of the two dimensions narrows the mutant down to one of 10 lines. The advantages of this population for reverse genetics are that (1) it is well-characterized, (2) seeds from individual lines are stored in a manner that facilitates various pooling strategies and subsequent identification of the mutant, and (3) seeds and DNAs are publicly available through the Arabidopsis Biological Resource Centers. The ability to isolate specific mutants or genes utilizing T-DNA mediated gene tagging in other species remains impractical.
