Seed Details

Primary transformants were selected in soil using Basta resistance.  Seed from individual T-1 plants was collected directly into 96-well plates.   Each well contains T-1 seed collected from a single T-1 parent plant.  Our entire collection of 63,000 lines is stored in 657 96-well plates.  Deep-well 96-well plates were used that have a capacity of 2 mls.

In order to streamline the mapping of T-DNA inserts in our population we developed a method for rapidly isolating genomic DNA directly from dry seeds.  This process allows us to obtain PCR-ready DNA from a small sample of ca. 50 T-1 seeds.  The sampling of 50 seeds from each well of a 96-well plate is performed simultaneously using a simple device, making it possible to collect all 96 samples in less than two minutes.

TAIL-PCR is then used to amplify PCR products that contain Arabidopsis genomic DNA that flanks the T-DNA insertion site.  These flanking sequences are used to precisely map the locations of the T-DNA insertions in the genome.

Because the seed that we send out to people comes from the same T-1 archive that was used for TAIL-PCR, problems associated with growth of the lines through a second generation are avoided.  In rare cases where we run low on a particular line, then seed from that line will be bulked-up.