Esterase and peroxidase zymograms from barley ( Hordeum vulgare L.) callus as a biochemical marker system of embryogenesis and organogenesis

Abstract

Based on visual appearance three different tissue types could be distinguished in callus of Hordeum vulgare L.: soft pale callus, compact pale callus and compact green callus. Soft pale callus contains highly vacuolated and enlarged cells. Compact callus, pale as well as green, consists of hard nodules with small thin walled cells. Esterase (EC 3.1.1.2) and peroxidase (EC 1.11.1.7) isoenzyme patterns were analyzed in the different types of barley callus, before any sign of differentiation was visible. The esterase system is very sensible for detection of embryogenesis before somatic embryos are formed. Peroxidase indicates both leaf and root morphogenesis before primordia can be seen. Transfer of callus on regeneration medium with lower auxin content confirmed the date obtained after enzymatic analysis, although lower percentages of plant regeneration were obtained, than those predicted after enzymatic analysis due to necrosis and suboptimal culture conditions. The results of the isozyme analyses further confirm that no difference should be made between hard green and hard pale callus. Based on the enzymatic data, it was concluded that soft callus and both types of hard callus display a low capacity of organogenesis (2–3%). The embryogenic capacity is much higher in pale and green hard callus (43–48%) compared to soft pale callus (5%). To optimize the in vitro culture of barley, it is useful to make a selection for the hard parts of the callus while eliminating the soft callus tissue.