Establishment and biochemical characterization of tamarillo (Solanum betaceum Cav.) embryogenic cell suspension cultures
Alves, A.; Caeiro, A.; Correia, S.I.; Verissimo, P.; Canhoto, J.
In Vitro Cellular and Developmental Biology. Plant 53(6): 606-618
Somatic embryogenesis (SE) is an important biotechnological tool with great potential for large-scale cloning. In Solanum betaceum Cav. (tamarillo), embryogenic (EC) and non-embryogenic (non-EC) cells can be obtained from the same explant on auxin-containing medium, making this system ideal for the evaluation of biochemical changes occurring during embryogenic induction. Liquid cultures offer additional possibilities for the analysis of factors controlling SE induction, and the main objectives here were the establishment of cell suspensions and the characterization of the extracellular protein profiles in EC and non-EC cultures. Growth kinetics of liquid cultures, starting with different amounts of EC or non-EC callus or with different weight per volume ratios, were analyzed. Embryogenic suspension cultures were efficiently established starting with 40 mg of cells in 20 mL of liquid medium. Mass spectrometry and fluorometric techniques were employed to identify extracellular proteins, their hydrolytic activity, and the main classes of proteases secreted into the media of EC or non-EC cultures. Extracellular protein profiles revealed quantitative and qualitative differences between EC and non-EC suspension cultures, mainly for several hydrolytic enzymes, such as glucanases and xylanases. Proteolytic activity analysis found serine proteases, aspartic proteases, and metalloproteases in EC cultures, whereas serine proteases were dominant in non-EC lines. For the first time, a protocol for the growth of tamarillo EC and non-EC suspensions was achieved. Moreover, the comparison of protein profiles between EC and non-EC lines showed pronounced differences in the proteolytic and glycolytic enzymes secreted.