To establish an experimental system to identify mutants and to clone corresponding genes we have been producing enhancer-trap lines of rice using vectors based on a maize transposable element Ac/Ds. We utilized enhancer-trap vectors, which consist of two plasmids, developed by Fedoroff and Smith (1993) with a modification (Fig. 1A, B). One is a vector containing an Ac transposase (AcTPase) gene driven by a CaMV 35S promoter (35S-AcTPase) and a bialaphos resistance gene in a T-DNA region (Fig. 1B). The other is a vector containing Ds between a 35S promoter and a coding region of a chlorsulfuron resistance gene in a T-DNA region (Fig. 1A). The Ds contains a GUS coding sequence with a minimal promoter and a hygromycin resistance gene. Thus, the bialaphos resistance and the hygromycin resistance can monitor existence of the AcTPase gene and the Ds, respectively. Furthermore, the chlorsulfuron resistance can monitor excision of the Ds from an original position in the vector.

We generated 263 transgenic rice lines by Agrobacterium-mediated transformation with the Ds vector, and 42 lines that harbor a single copy of the Ds were selected by Southern blot analysis. Six lines that harbor the 35S-AcTPase vector were also generated. We crossed 6 lines of the 35S-AcTPase with 4 lines of the Ds, and somatic excision in leaves of F₁ plants was examined by PCR. Because primers 1 and 5 for the PCR were set up to flank the Ds (Fig. 1A), a band of about 600 bp could be amplified if the excision of the Ds occurred, and a 6-kb band could be amplified if the excision of the Ds did not take place (Fig. 1C). The result is summarized in Table 1. The somatic excision was detected in 8 combinations out of 17 examined. Ds in line E029 did not transpose in any combinations with the 35S-AcTPase lines and the 35S-AcTPase in line A015 did not

support the transposition of Ds in any of the Ds lines.

We next examined frequencies of germinal transposition. DNA isolated from leaves of F₂ plants were used as templates for PCR. Three combinations of 6 primers were used. The combination of primers 3 and 4 monitors existence of the Ds (Fig. 1A). The combination of primers 1 and 2 monitors existence of the untransposed Ds (Fig. 1A). The third primer combination, which amplifies a part of an endogeneous OSH1 gene, was used as a positive control of the PCR. The F₂ plants which contain the Ds but not untransposed Ds were selected as transposants (Fig. 2, lanes 5 and 14, for example). We screened 8670 F₂ plants, and 620 (about 7%) were judged to be transposants. The F₁ plants which showed somatic excision in the leaves produced the germinal transposants at high frequency (15% of the F₂ plants), while the F₁ plants which did not show the somatic excision produced germinal transposants at low frequency (1%). The frequency of the germinal transposition was greatly different from panicle to panicle, suggesting that Ds tends to transpose during panicle development. These results indicate that the enhancer-trap vectors used in this study are applicable to rice for screening various types of transposants.

Reference

Fedoroff N.V. and D.L. Smith, 1993. A versatile system for detecting transposition in Arabidopsis. Plant J. 3: 273-289.