Tissue culture triggers chromosome alterations, amplification, and transposition of repeat sequences in Allium fistulosum

Structural alterations in nuclei and chromosomes of cells derived from callus culture of Allium fistulosum have been studied with fluorescent in situ hybridization (FISH) using 5S ribosomal DNA (rDNA), 45S rDNA, and 375-bp repeat probes. A high frequency of chromosome abnormalities was found to be caused by the loss of telomere-located 375-bp repeats, chromosome fusion, and subsequent breakage–fusion–bridge cycles. Products of chromosome fusions and monocentric and regularly shaped chromosomes showed additional 375-bp repeat and 45S rDNA clusters at unusual sites, suggesting dynamic copy-number changes and transposition of these repeats. Southern hybridization revealed no differences in the 375-bp repeat and 45S rDNA repeat array order or the degree of methylation between DNA isolated from leaves or tissue-culture cells. In addition, protruding, spike-like structures positive for 375-bp repeats were identified on the surface of different-sized nuclei. Transmission electron microscopy analysis revealed the accumulation of densely packed chromatin within spike-like structures. Because root calyptra cells showed similar structures, it is likely that heterochromatic spike-like structures are a feature of nondividing cells at the onset of programmed cell death.