Functional Genomics
Supported by the NSF Plant Genome Program, we are in the process of constructing a transgenic transposon-tagging genetic resource to meet the functional genomics needs of the maize community. The resource is designed to mobilize a modified Ds element from different starting sites in the genome. We made 19 different constructs to test the activity of engineered Ds transposons, 16 based on pBS or pUC plasmids for use in biolistics experiments and 3 based on the pTF102 binary vector plasmid for use in Agrobacterium transformation experiments. The mutable alleles were engineered versions of either bz-m2 or c1-m2.
1.
Biolistics
From 68 shooting experiments, we produced 271 independent
transgenic events, regenerated over 1000 plants carrying
the constructs, and tested transposon activity by
crossing to appropriate Ac stocks. Of the 271 events, 74
transformants were somatically active. Only 1 of the 19
somatically active bz-m transformants produced Bz'
germinal revertants, whereas 20 of the 55 somatically
active c1-m transformants produced C1' germinal
revertants (Fig. A above), which are potential sources of
transposed Ds elements. The C' revertants were analyzed
molecularly for the presence of trDs elements. Of 210 C'
germinal revertants analyzed, 99 carried new trDs bands,
a result consistent with the one-half fraction seen in
other genetic screens based on transposon excision.
2. Agrobacterium
From 10 transformation experiments, we produced 46
independent transgenic events. Most c1-m transformants
generated by Agrobacterium transformation show kernel
spotting when crossed to c1 wx-m7(Ac) testers. The
spotted phenotype resembles that of the native c1-m2
allele and segregates 1:1 (Fig. C above), suggesting
integration of the transgene(s) at a single Mendelian
locus. Southern Blot analysis confirmed the presence of 1
to 2 T-DNA copies per transgenic line. We are currently
characterizing the T-DNA integration sites in the
germinally active c1-m transformants.