Abstract

Rudo Ngara, Jasper Rees and Bongani K. Ndimba*

This study describes the establishment of sorghum cell suspension culture system for use in proteomics studies. Friable sorghum callus was initiated from young shoots under completely dark conditions on MS medium supplemented with 3 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2.5 mg/L 1-naphthaleneacetic acid (NAA). Additionally, sorghum cell suspension cultures have been initiated from the friable callus masses in liquid medium with the same composition as the callus initiation medium. Total soluble proteins (TSP) and culture filtrate (CF) proteins were extracted from the cell culture system and solubilised in urea buffer (9 M urea, 2 M thiourea and 4% CHAPS). Both onedimensional (1D) and two-dimensional (2D) gel analysis of these two proteomes show that the TSP and CF proteomes have different protein expression profiles. The sorghum TSP proteome, which is highly complex, is best resolved when separated on large format, 18 cm, pH 4 - 7 isoelectric focusing (IEF) immobilised pH gradient (IPG) strips. On the other hand, the sorghum CF proteome (secretome) is less complex with most proteins being resolved on mini format, 7 cm, pH 3 - 10 IPG strips. Furthermore, narrowing down the pH range from 3 - 10 to 4 - 7 for the CF proteome resulted in improved protein spot resolution.