TISSUE CULTURE-INDUCED DNA METHYLATION VARIATION IN MAIZE

Twenty-one progeny lines derived from tissue cultures of two embryo so urces of maize inbred strain A188 were examined for DNA methylation ch anges. Total DNA was cut with the isoschizomers Hpa II and Msp I and p robed with 18 single-copy Pst I genomic clones and two cDNA clones. Ei ght of these probes could detect both increases and decreases in methy lation. With these probes 39 % of the families were found to contain a n altered methylation pattern. All changes represented a decrease in m ethylation. The other 12 probes could detect only increases in methyla tion; no methylation variation was seen with these probes. Fifteen per cent of the methylation changes were homozygous in the original regene rated plant. Changes were stably inherited upon two generations of sel f-pollination. No sequence variation was observed in Msp I-digested DN A from the same 21 progeny lines. Certain probes detected methylation changes much more often than others. Our study provides evidence that demethylation occurs at a high frequency and could be an important cau se of tissue culture-induced variation. Occurrence of the frequent hom ozygous alterations in original regenerated plants implies a non-rando m mutational mechanism.