We are trying to understand the basis of the instability of Sn:bol3, one of the Sn geographic alleles (MNL, 1988). In fact the progeny of individuals selected for strong red scutellar node and mesocotyl (referred to as Sn-s) consist of seedlings with parental phenotype as well as seedlings with significantly lower levels of pigmentation (referred to as Sn-w) and arising with a high frequency (from 20 up to 60% of the total progeny). Sn-s selections keep showing instability in succeeding generations while Sn-w selections never revert to the original phenotype.
Southern analysis of genomic DNA, digested with SstI and HindIII and probed with the 600 bp R-nj fragment (as reported in MNL 1988), shows the same hybridization pattern in Sn-s and Sn-w, thus suggesting that no large DNA rearrangements are involved in this event.
Therefore we postulated that Sn:bol3 instability could be correlated to DNA modification. Accordingly we treated Sn-w derivatives with the deoxycytidine analog 5-aza-2'-deoxycytidine (Aza) in order to see if the analog is capable of inducing a phenotypic reversion of Sn-w to Sn-s. Homozygous Sn-s and Sn-w seeds were germinated in darkness in the presence of the analog. Five days after germination they were exposed to light for 4 hours and the anthocyanin content of the scutellar node was then determined. The results obtained (Table 1) show striking differences in the level of pigmentation. The Sn-w derivatives treated with Aza show a dramatic increase in their pigment content, about 70 fold increase, when compared to the untreated ones. The effect of the analog is evident also in homozygous Sn-s seedlings, where a 4-5 fold increase in pigment content is observed. Furthermore a comparison between the values obtained in the treated Sn-w versus untreated Sn-s tissues show that Sn-w derivatives not only revert to the original phenotype but show a higher pigment content. These results thus suggest an involvement of DNA modification in Sn instability. Preliminary results of Southern blot analysis of genomic DNA from treated and untreated plants, digested with methyl-sensitive enzymes, seem to confirm this view.
Table 1. Effect of 5-aza-2' deoxycytidine (aza) on anthocyanin accumulation in scutellar node of Sn-s and Sn-w strains.
C. Tonelli, G. Consonni, G. Gavazzi and A. Viotti
1Instituto Biosintesi Vegetali, C.N.R.
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