TISSUE CULTURE-INDUCED DNA METHYLATION POLYMORPHISMS IN REPETITIVE DNA OF TOMATO CALLI AND REGENERATED PLANTS

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has be en proposed as a mechanism that may explain at least a part of these c hanges. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, a t MspI/HpaII sites around five middle-repetitive sequences. Although t he methylation of the internal cytosine in the recognition sequence CC GG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and ca llus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regen erated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, ind icating heterozygosity of the regenerated plants. The data are interpr eted as indicating that a HindIII site becomes methylated in callus ti ssue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.