Rice Genetics IV - IRRI books - International Rice Research Institute

ence of a single excision footprint in all the regenerants from a single T-DNA insertion(Greco et al, submitted) suggests that transposition must have begun from anunreplicated site. The mechanism of Ac amplification we propose is outlined in Figure2 and is potentially useful for generating genotypes with multiple transposons(Fig. 1) that can reach genome saturation quickly.To compare the transpositional activity between transformants, a parameter termed“independent transposition frequency” (ITF) given by the frequency of unique transpositionevents among the total inserts in a set of related plants (regenerants or seedprogeny) was estimated. The somatic ITF calculated over three populations ofregenerants varies between 50% and 65%. Transposed Ac insertions that were presentin only a single plant were considered as unique late events that were useful for producingdifferent insertions in the progeny.In contrast to the active transposition in regenerating calli, leaf samples of matureplants revealed a stable transposon insertion pattern. The individual progeny againrevealed new insertions with an ITF between 15% and 50% in different lines, suggestingthat transposition took place prior to gamete formation. This indicates a biphasictransposition pattern: an active phase during regeneration from the callus as well asthe pregametic cells and a stable phase in the mature plant.TranspositionReplicationUnreplicated T-DNA siteUnreplicated target siteTranspositionTranspositionReplicationUnreplicated target sitesFig. 2. Ac amplification during replication in one cell. The mechanism of Ac amplification proposedto explain the results of Ac (Ac designated as triangle) transposition observed. The hightransposase activity mediated by the enhancer adjacent to the Ac promoter in the initial cellafter transformation and before further cell division triggers an early excision event from theunreplicated T-DNA site (shaded box) that is revealed by the occurrence of the same excisionfootprint (open star on chromosome site) in every regenerant. In the same cell cycle, theexcised Ac reinserts into a second site, which then replicates to generate a second Ac copy inthe newly replicated strand. In the continued presence of the high Ac transposase activity inthe same cell, secondary transposition events of both Ac copies result in two new unreplicatedpositions that get duplicated following replication, giving rise to four copies at the two chromosomalpositions. The high transposase activity mediates a third cycle of transposition andreplication that produces four new Ac chromosomal positions (denoted by different shadedlines) in the cell.270 Greco et al