Rice Genetics IV - IRRI books - International Rice Research Institute

Whole-plasmid DNAClean fragmentBombard callusRegenerate plantsSouthern blotPlasmid DNA: can generatecomplex integration patterns,with loss of expressionstabilityClean fragment: predominantlysimple integration patterns,stable transgene expressionFig. 4. Clean DNA transformation. Clean DNA is the linear minimaltransgene expression cassette, cleaved from the plasmid usingrestriction enzymes or generated by polymerase chain reaction. Incomparative experiments, clean DNA generates simpler integrationpatterns and a greater proportion of plants with stabletransgene expression, even though overall transformation frequenciesare similar using the two methods.this issue by studying a single transgenic rice line containing a single copy of a constructcomprising three heterologous transgenes: hpt, gusA, and bar (Fu et al 2000a).By concentrating on this line, we eliminated variation caused by different positioneffects and different transgene copy numbers, since the transgene locus was identicalin structure in each plant we analyzed. Furthermore, because there was only a singlecopy of the locus and because none of the transgenes were homologous to endogenousrice genes, we eliminated the possibility that ectopic pairing could influencetransgene expression.Despite these precautions, we found that silencing still occurred de novo in severalR 1 , R 2 , and R 3 plants. In all cases, silencing was associated with DNA methylationof the silenced locus, but in some cases this was heritable through meiosis and in460 Christou et al