Rice Genetics IV - IRRI books - International Rice Research Institute

Number of loci36%224%170%010 20 30FrequencyFig. 3. Estimation of T-DNA insertion loci. The number of geneticloci was estimated by scoring hygromycin-resistant progeny.single locus. The result revealed that two lines did not contain any DNA sequencesbetween the T-DNAs (Fig. 1B). The remaining four lines carried 6 to 488 bp of fillerDNA. Interestingly, the 488 bp of the longest filler DNA in the B1558 line was foundto be a portion of the gus gene. A DNA gel-blot analysis confirmed that the B1558line had one more copy of gus than hph. Such a partial T-DNA was previously reportedfrom dicots such as tobacco (Krizkova and Hrouda 1998). It appears that theformation of repeated T-DNA copies might result from co-integration of several intermediatesinto one target site.It has previously been reported that a majority of the T-DNA insertions occurwithin the right border at a specific locus (reviewed in Tinland 1996). To examinewhether the same was true for our tagging lines, the junction regions between ricegenomic DNA and the T-DNA right border were sequenced (Fig. 1C). The sequencingresults revealed that the boundaries in most of the rice lines did not correspond tothe T-DNA nicking position found in Arabidopsis and tobacco transgenic plants. Indicot species, most T-DNAs were nicked after the first or second base of the rightborder. In our tagging lines, five showed nicking positions similar to those ofArabidopsis and tobacco. However, the most frequent junction point (11 out of 32lines) was after the third base of the right border. In seven lines, the junction was atthe boundary between T-DNA and the right border. The remaining nine lines showeddeletion of one to 12 bases of T-DNA. It was previously reported that two of threeright boundaries in transgenic rice plants and four of ten in transgenic maize plantscarried three bases originated from the right border (Hiei et al 1994, Ishida et al 1996).Expression of the gus gene in transgenic rice plantsTo evaluate the efficiency of the gene trap system, the gus expression pattern wasexamined from various organs of primary transgenic plants transformed with pGA2144.We have analyzed GUS activity in the leaves and roots from 5,353 lines, in matureflowers from 7,026 lines, and in developing seeds from 1,948 lines. The results re-Generation of T-DNA insertional tagging lines in rice 257