Rice Genetics IV - IRRI books - International Rice Research Institute

appealing in regard to our efforts to combat the RWW in its larval stages. However, instudying more than 100 plants independently transformed with an RCg2/(β-glucuronidase)uidA construct (pYXB, Fig. 1C), we have found that less than 20% yieldGUS expression and that few of these plants express strongly (Table 1, Fig. 3), eventhough Agrobacterium-mediated transformation was used (Dong et al 1996) to generateplants that have simple, low-copy-number integration patterns. We have confirmedthat the majority of the plants are indeed silenced by observing reactivation of GUS inmature roots (Fig. 3B) and seedlings treated by AzaC (50 mg L –1 ). The observation ofbrief reactivation of GUS activity from the RCg2-uidA in germinating seedlingstransgenic for pYXB also confirmed silencing of RCg2-uidA. Although mature rootsexhibited weak GUS expression, many of the young seedlings showed strong expression,indicating meiotic resetting of silencing. However, activity decayed after about5 to 7 d. The reactivation by AzaC and meiotic resetting of RCg2/uidA are not uniformamong the lines or even throughout individual plants, indicating that various silencingmechanisms may operate and that the chromosomal position of the transgene mayaffect the silencing processes. We have established fluorescent in situ hybridization(FISH) techniques for rice transgenes (Fig. 3D) and hope to gain insight into thepossibility that position affects the susceptibility of a transgene for silencing.Table 1. A summary of transgene expression analysis of YXB plants. More than 100 independentrice lines were analyzed for the presence and expression of mUbi1/bar and RCg2/uidA.More than 80% of the YXB plants showed silencing of RCg2/uidA while the flanking mUbi1/barand 35S/hpt genes remain active, indicating that the silencing is highly efficient and targetedspecifically to RCg2/uidA.ProteinExpressingTransgene analysisNonexpressing aGUS Strong (8) Weak (19) Gene* present (55) Absent (11)49a, 68, 69a, 56–59, 61, 79, 122, 2, 12, 19, 24, 25, 33, 37, 1, 32, 50,91–93, 98, 126–128, 130, 135, 44, 49b, 51–52, 54, 55, 64, 76, 78,138 147, 149, 151, 153, 60, 62, 63, 65–66, 69b, 86, 96, 105,156–158 71–73, 75, 80, 82, 84, 121, 12988–90, 100–102, 104, 106,108–109, 114–115, 119,123–125, 131–134, 136–137,139–141, 143–146BAR Resistant (74) Gene* present (7) Absent (12)2, 12, 24, 25, 33, 37, 44, 49a, 19, 62, 76, 96, 101, 136, 1, 32, 50,49b, 51, 52, 54–61, 63, 66, 68, 149 64, 65, 78,69a, 69b,71–73, 75, 79, 80, 82, 86, 105,84, 88–93, 98, 100, 102, 104, 109, 129,106, 108, 114, 115, 119, 121–128, 132, 134130, 131, 133, 135, 137–141,143–147, 151, 153, 156–158a* = intact transgene.470 Hall et al