A tissue culture system facilitates examination of gene expression during breakage of vegetative dormancy in the potato tuber shoot apical meristem

While the shoot apical meristem's role during dormancy emergence in seeds is relatively well-understood, molecular factors governing vegetative endodormancy and sprouting in tuberous plants have not been well studied. A microtuber culture system was developed in order to study dormancy emergence in the shoot apical meristem of the potato (Solanum tuberosum). Microtubers were induced from subcultured shoot internodes, grown for 9 weeks, then harvested. The microtubers were hardened at 20° for 2 weeks then stored in the dark at 4°C. Subsequently, at 4-week intervals, microtubers were transferred to 20°C in the dark for 7 days. Meristem length was measured prior to RNA extraction. Two genes, SHOOTMERISTEMLESS (STM) and WUSCHEL (WUS), are known to regulate the development of the shoot apical meristem in Arabidopsis thaliana. Primers for quantitative PCR (qPCR) were designed to amplifty putative potato STM and WUS homologs using expressed sequence tag (EST) sequences obtained during a basic local alignment search tool (BLAST) search of known petunia (Petunia hybrida) and tomato ( S. Lycopersicum) STM and WUS sequences. Primer pairs for reference genes were obtained from the literature. Using qPCR we measured the expression level of STM normalized to two potato meristem reference genes, L2 and ef1-alpha. L2 expression increased 1.5 to 6 fold during the post harvest period while that of ef1-alpha expression was between 1 and 1.4 fold the levels found in dormant meristems immediately after harvest. By contrast, STM expression increased from 0.17 to 0.75 fold of week 2 values 7 weeks after harvest from 17.4 to 42.5 fold 11 weeks after harvest and increased further thereafter. The meristem length showed a statistically significant increase from 0.23 (+/- 0.01) mm to 0.33 (+/- 0.07) mm (mean +/- Standard Error) length between 7 and 11 weeks after harvest. Taken together, this data suggests dormancy break of shoot apical meristems in this microtuber culture system occurs between 7 and 11 weeks after harvest. This system will be used in the study of the molecular events occurring during dormancy break in the potato shoot apical meristem.