MAINTENANCE OF THE INDUCED HYPOMETHYLATED STATE OF TOBACCO NUCLEAR REPETITIVE DNA-SEQUENCES IN THE COURSE OF PROTOPLAST AND PLANT-REGENERATION

Previously, we established experimental conditions allowing us to indu ce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezdek et al. 1991, Pl anta 184, 487-490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-diff erentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 10(5) copies in the genome, can escape de-novo methylation.