II.49 Callus cultures of Hordeum vulgare: Initiation, growth and organogenesis.
H.M. Wilson, B. Foroughi-wehr, G. Mix, and H. Gaul. Abteilung für Pflanzengentik der Gesellschaft für Strahlen- und Umweltforschung, 8059 Grünbach 1, ub. Erding/ Obb. West Germany."R"
Regeneration of plants from callus or cell suspension cultures derived from somatic explants has been achieved from many species. Cereals, however, have proved relatively recalcitrant (see Sheridan 1975) though in the case of Hordeum vulgare a method for obtaining rapidly proliferating callus tissues with subsequent differentiation of plantlets have been described (Cheng and Smith 1975). These authors used apical meristems dissected from one-week-old seedlings of the varieties "Himalaya" and "Mari". We have repeated this work with the varieties "Mutina", "Edelmut", "Hilde" and "Nackta". Although callus growth on the initial explants was good, excision and transfer of this callus to fresh medium rarely resulted in the production of large quantities of growing material. However, in those cases where callus growth of a type allowing repeated subculture was initiated, it was possible to stimulate the production of shoots and roots by omitting all growth regulators from the medium (after Cheng and Smith, 1975).
In some circumstances it may be an advantage to obtain callus from explants of older plants and thus we have attempted to initiate callus from the nodes of Hordeum vulgare. Nodes were sterilised by immersion for 30 min. in 10% Calcium hypochlorite solution, containing a few drops of detergent to facilitate adequate "wetting". This was followed by five washes in sterile distilled water and the nodes were then bisected longitudinally and placed with the cut surface directly on the culture medium. Alternatively nodes may be dissected prior to sterilisation and then dissected again before inoculation. The latter method results in fewer infected cultures but also in smaller explants. The basal culture medium used was that of Linsmaier and Skoog (1965). While initiation and relatively good callus growth occurred on many of the media tried, when the callus tissues were excised from the node explant and transferred to fresh medium, growth invariably ceases. It made no difference when the callus tissues were allowed to remain with the explant on the same medium, or, were transferred together with the explant to fresh medium. Various medium constituents were tested including the hormones IAA, NAA and 2,4-D (1,0-20 mg/l), the cytokinins benzyladenine purine and kinetin (0,2-20 mg/l), and gibberelline (2,0-10 mg/l). The effectiveness of NH4NO3, inositol and thiamine was also examined. The plant growth regulators were tested in combinations of two or three together in the same medium and the cultures were incubated at 25°C either in the dark or with a 16 hr. photoperiod. The most effective single constituent tested for the initiation of callus appeared to be 2,4-D.
The media used successfully by Cheng and Smith (1975) for apical meristem explants proved ineffective when nodes were used and tissues capable of repeated subculture were not obtained. Thus it appears that the source of the initial explant is of some importance in this case. It is clear that the node explants are supplying some factor(s) which is essential for callus growth and that once this endogenous supply is interrupted or exhausted, growth is terminated. Attempts to identify the substance(s) involved are in progress.
References:
Cheng, T.Y., and H.H Smith 1975 Organogenesis from callus culture of Hordeum vulgare. Planta 123, 307-310.
Linsmaier, E.M., and F. Skoog 1965. Organic growth factor requiremts of tobacco tissues culture. Physiol. Plantarum 18, 100-127.
Sheridan, W.F. 1975. Plant regeneration and chromosome stability in tissue culture. pp. 263-295 in Genetic Manipulation in Plant Material. Proc. NATO International Summer School, Liege 1974. Ed. L. Ledoux. Plenum Press.