METHYLATED DNA CHANGES ASSOCIATED WITH THE INITIATION AND MAINTENANCEOF VITIS-VINIFERA IN-VITRO SHOOT AND CALLUS-CULTURES - A POSSIBLE MECHANISM FOR AGE-RELATED-CHANGES

Tissue culture technologies are an important aspect of the genetic mod ification of grapevine (Vitis vinifera). The molecular basis of this p henomenon is not well understood, however, the extent of DNA methylati on is recognised as a factor in the control of gene expression. This s tudy explores the possibility that DNA methylation may have a role in grapevine culture responses. DNA methylation profiles were constructed for Vitis vinifera, cv. Sultanina, during plantlet micropropagation, callus induction and proliferation. Methylation of genomic DNA and rib osomal RNA genes (rDNA) was found in glasshouse-grown plants, micropro pagated plantlets and callus cultures. An analysis of rDNA showed that glasshouse-grown plants had 74.6% of the recognition sequences for Hp a II methylated at the internal cytosine position CmCGG, whereas 7.7% of the recognition sequences appeared to be methylated at the external cytosine (mCmCGG). The rDNA profiles of micropropagated subcultures S -0 (initial subcultures) and S-4 (fourth subcultures), representing on e year of in vitro growth, showed that the percentage of recognition s equences containing a methylated external cytosine increased from 7.7% in glasshouse-grown plants to 64.5% for S-0 and 72.5% for the S-4 sub culture. The implications of these findings for the in vitro manipulat ion of grapevine used in genetic modifications are discussed.