mRNA localization in in vitro grown microtubers of potatoas a tool to study starch metabolism

In vitro grown microtubers were used to investigate whether in situ hybridization is a feasible technique to study expression of genes involved in starch metabolism in potato (Solanum tuberosum L.). Single node cuttings containing an axillary bud were placed on a tuber-inducing medium. Microtubers were harvested 7 d after culture initiation, and were subsequently prepared for in situ hybridization with antisense RNA probes derived from cDNA clones encoding for sucrose synthase (EC 2.4.1.13), granule bound starch synthase (EC 2.4.1.21) and starch branching enzyme (EC 2.4.1.18). Tissue specific expression can be monitored with the use of dark field microscopy at low magnification. This technique showed that the sucrose synthase gene is expressed in the tuber, but not in the apical meristem or the stolon on which the tuber was formed. Bright field microscopy allows the visualization of mRNAs at the subcellular level. mRNAs encoding for granule bound starch synthase and starch branching enzyme were accumulating primarily around the amyloplast. In contrast, the mRNAs encoding for sucrose synthase did not show a preferential distribution. These results demonstrate the feasibility of in situ hybridization for potato tubers, and suggest an additional level of regulation in starch metabolism.