Variation in potato microplant morphology in vitro and DNA methylation

Abstract

Heterotrophic and autotrophic culture in agar and in polyurethane foam, the latter used as an alternative tissue support to agar, resulted in potato microplants with different in vitro morphologies. The microplants were visually characterised in terms of their relative developmental maturity, by comparing the respective leaf shapes in vitro with ontogenetic differences in leaf shape in glasshouse-grown potato plants. Cytosine methylation in the DNA of microplants of the different morphologies was determined using a method based on the AFLP technique but employing methylation-sensitive restriction enzymes (MSAP analysis) to test the hypothesis that DNA methylation could be used to characterise differences in microplant development in vitro. In three of the four treatments there was a good correlation between the visual assessment of relative morphological maturity and DNA base methylation levels. In these microplants there was increased DNA methylation in the leaves with mature leaf morphology represented by a decreased number of restriction fragments. The fourth in vitro morphology had the most juvenile leaf shape but did not have the predicted level of DNA methylation, having a relatively low number of restriction fragments. Subtraction analysis was used to discriminate the fragments that were unique to the juvenile and mature in vivo leaf morphologies. Comparison of the fragment patterns from the microplants with the latter reference profiles, confirmed the relationship with the total DNA methylation as detected by MSAP analysis, that is, the number of common fragments with the juvenile or mature in vivo leaf profiles, respectively. However, none of the fragment profiles, while sharing some common bands at random, was identical to any other; or to that of either the juvenile or mature in vivo leaf. The anomalous relationship of the microplants with most juvenile leaf shape and highest DNA methylation was confirmed. The measurement of DNA methylation in in vitro plants is discussed in the context of the development of a method to assess the quality of microplants produced by different in vitro protocols.