| Subculture of Wheat Callus Wheat callus derived from filament tissue was grown on 27 different combinations culture media to test for differences in growth rate. The media tested were: I, Linsmaier and Skoog; II, Linsmaier and Skoog, with the basal salts modified to contain 5312 mg/l KNO3, 330 mg/l NH4N03, and 167.5 mg/l CaCl2 (personal communication, C.E. Green, Univ. of Minn.); and III, White's modified according to SHARP et al. (1972). Each of the media was supplemented with either 1 g/l casamino acids, 0.5 g/l of yeast extract or both 1 g/l casamino acids and 0.5 g/l of yeast extract. Sucrose was included at 2, 4, or 6%, and all of the media contained 4 mg/l of 2,4-D. In most cultures, a cream-colored callus with a raspberry-like appearance was formed on the medium. Although callus survived and proliferated on all of the media, there was considerable difference in growth rate. The greatest increase in size occurred on the modified Linsmaier and Skoog's medium containing casamino acids only (IIa). Only a slight difference was apparent in the effect of sucrose concentration in this medium, with 4% sucrose appearing to be superior. The substitution of yeast extract for casamino acids (IIb) or its addition to the medium containing casamino acids (IIc) resulted in a marked inhibition in growth and the callus being yellow to tan in color. The second-best culture medium was the regular Linsmaier and Skoog's medium containing casamino acids only (Ia). Little effect of sucrose concentration was evident, but the inhibitory effect of yeast extract was clearly evident and affected callus growth and coloration in the same way as described above. Wheat callus growing on the modified White's medium grew more slowly than on the other media, but showed slowest growth when only casamino acids were present. Inclusion of yeast extract alone (IIIb) was superior to casamino acids alone (IIIa), and the inclusion of both supplements (IIIC) gave the best growth response. There was little effect of sucrose concentration on growth. Three conclusions regarding growth rate of wheat callus could be clearly drawn from this experiment. First, Linsmaier and Skoog's medium was superior to the modified White's medium; second, the inclusion of yeast extract in Linsmaier and Skoog's medium was clearly inhibitory alone or in combination with casamino acids, while its inclusion in the modified White's medium alone or in combination with casamino acids was clearly stimulatory; and third, the inclusion of sucrose at 2, 4, or 6% concentration in these media had only slight effects on growth rate. Wheat callus, originally derived from filament, has been repeatedly subcultured in liquid basal medium containing 2% sucrose. Under these conditions the callus grows as white- to cream-colored clumps which continually fragment and thus remain a few millimeters or less in diameter. The callus remains undifferentiated in this 2,4-D-containing medium, but upon transfer to basal medium containing 2% sucrose and 4 mg/l of NAA and lacking 2,4-D, the callus differentiates into hundreds of roots, which rapidly elongate and form a filamentous mass radiating from the central callus clump. These roots remain white except for some green pigmentation appearing in the vascular region which occupies the core of the roots. No stem or leaf differentiation has been observed. |
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