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The frequency variation of highly repeated (HR) DNA sequences was studied in plants regenerated through in vitro culture of macerated vegetative apices of Pisum sativum and their progenies. Feulgen cytophotometry showed that regenerated plants (R1) have 6-7% more DNA per nucleus than control plants; this difference is maintained in the subsequent generation (R2). Slot-blot analyses using isolated highly repeated sequences as probe indicated that an increased frequency of these sequences occurs in regenerated plants and their progenies. These results were confirmed by a series of experiments: (a) metaphase chromosomes were longer in R2 than in control plants; (b) cytophotometric analyses of chromatin structure after Feulgen-staining showed that condensed chromatin is more represented in R2 than in control plants; and (c) thermal denaturation of isolated HR sequences indicated that a new HR family appears in R1 and is maintained in R2 plants. These results suggest that, in this species, the DNA extrasynthesized during in vitro culture is, at least in part, integrated in the chromosomes and transmitted to the progeny.
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