Identification of Gene Expression Patterns During Somatic Embryogenesis in Potato

This investigation was aimed at studying global gene expression during the process of somatic embryogenesis in potato using internodal segments (INS) as explants. The INS explants from 4 to 6 week old potato cv. Desiree in vitro cultures were cultured on somatic embryogenesis induction for 2 weeks (stage-I) and expression/regeneration modified MS media for 3 weeks, respectively with (stage-II) and without (stage-III) 2, 4-dichlorophenoxy acetic acid (5 µM). The culturing frequency was 10 INS/petridish with 25 ml medium. The in vitro cultures were incubated at 19±1°C, 16/8 hrs light/dark cycles and 90 µmol/m2/s light intensity. To study the molecular events of SE as affected by the absence or presence of auxin, the INS explants were divided into 2 groups after the induction phase. One-half of the induced INS explants were transferred to auxin free medium to evoke SE, the other half were subjected to a prolonged induction phase. The total RNA from INS explants at these critical stages of SE progression was isolated using TRIzol (Invitrogen) as per the manufacturer’s instructions. The on-column DNase treatment and clean-up steps were performed using Qiagen RNase free DNase and RNeasy midi kits. The selected time points used for the RNA isolation were day 0 (freshly cut INS), day 14 (INS on induction medium) and day 21, 28 & 35 (INS on expression medium as well as from prolonged induced phase). Except day 0, where the whole INS were used for RNA isolation, all other time point RNA was isolated from the callused part of the INS distal end. Each time point was replicated 3 times. The number of explants for each biological replicate varied from 30 to 300 owing to variability in tissue availability and corresponding RNA yield from explants of each time-point. The entire process from tissue excision to RNA isolation was completed on the same day to avoid any unintended change in tissue condition by storage. Keywords: Direct comparison 28 hybs total