ISSN 2756-3707
International Journal of Histology and Cytology ISSN 2447-9535 Vol. 3 (10), pp. 287-292, October, 2016. © International Scholars Journals
Full Length Research Paper
Establishment of sorghum cell suspension culture system for proteomics studies
Rudo Ngara, Jasper Rees and Bongani K. Ndimba*
Proteomics Research Group, Department of Biotechnology, University of the Western Cape, Private Bag X17, Bellville, 7535, Cape Town, South Africa.
*Corresponding author. E-mail: [email protected]. Tel: +27 21 959 2468. Fax: +27 21 959 1551.
Accepted 07 November, 2015
Abstract
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 one-dimensional (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.
Key words: Sorghum, proteomics, callus, cell suspension cultures, total soluble protein, secretome.