PCR analysis of bisulfite-modified DNA to investigate developmental variation in micropropagated potatoes (Solanum tuberosum L.)

Bisulfite modification of DNA from potato leaves showing juvenile and mature leaf morphologies and from leaves of autotrophic and heterotrophic microplants showing different morphologies was carried out to deaminate cytosine residues while leaving 5-methylcytosine residues intact. RAPD analysis of the DNA was then performed using selective primers; firstly, with a 50-70% GC content and subsequently with a 50-70% AT content which contained CG or CA 3' ends. Three of the 4 primers used detected band polymorphisms in the juvenile versus mature leaves and all four primers detected band polymorphisms in the autotrophic versus heterotrophic in vitro leaf samples. These results indicate differences in methylation in the different paired DNA samples. The hypothesised increase in band numbers in the relatively less mature samples was confirmed for both the in vivo and in vitro leaf samples; a higher number of bands was amplified from the DNA of the leaves from heterotrophic cultures compared with those from autotrophic culture when the results for all primer sets were grouped, however, this trend did not apply to the results from all the individual primers. The application of this approach to the evaluation of in vitro protocols for potato micropropagation is discussed.