In situ PCR on plant material with sub-cellular resolution

Abstract

In situ PCR and reverse transcribed in situ PCR have been tested on leaves of sugar cane fixed in FAA, 4% PFA or 2% PFA+2.5% GA. In situ PCR amplification of the gene coding for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) was successfully performed following all three fixation protocols. As expected the PCR product was restricted to the plastids of all cells. Reverse transcribed in situ PCR was performed on rbcL mRNA and in this case the PCR product was restricted to the plastids of the bundle sheath cells. This is the first report of in situ PCR on plant material and only the second report of in situ PCR with sub-cellular resolution. In situ PCR on rbcL may prove to be a valuable positive control for future in situ PCR studies on plant material.