Exploring the use of cDNA-AFLP with leaf protoplasts as a tool to study primary cell wall biosynthesis in potato

An RNA fingerprinting study of potato leaf protoplasts was performed to explore its suitability for identifying candidate genes involved in primary cell wall biosynthesis. Microscopic analysis, using calcofluor white to stain cellulose, showed that the protoplasts generated a new cell wall in the first 18 h after transfer to a culture medium. Complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to visualise differential gene expression at five distinct time-points within these first 18 h. In vitro plants (with and without exposure to severe physical damage) served as controls. Around 8500 transcript derived fragments (TDFs) were visualised which showed varying expression patterns in the protoplasts and controls. In total 156 TDFs were isolated, sequenced and used to search for homologies. Over 50% of these TDFs showed homology to described genes, involved in several general plant processes. However, only one cell wall related TDF (a pectin esterase) was found. Our results showed that even though the protoplasts actively regenerate a new cell wall, this did not result in highly increased expression of genes involved in cell wall biosynthesis or modification. [Copyright &y& Elsevier]