Seedlings of Dioon edule Lindl. cultured aseptically on agar slants of modified White's medium at pH 5.7 failed to develop root nodules in light or darkness. Light inhibited primary-root elongation and secondary-root production and stimulated callus formation from a periderm-like tissue which developed in both primary and secondary roots.

Each treatment contained six individuals. Statistical significance of mean growth measurements was tested at the 5% level with Wilcoxin's signed ranks test which has virtually the same sensitivity as the student's t test (Langley, 1970). Horizontal lines below the x axis indicate significance as determined by the signed ranks test. Treatments without overlapping lines are significantly different. Where results were obviously different, statistical tests were not used.

The anatomy of light and dark-grown primary and secondary roots was examined by making transverse free-hand sections and staining them with 0.005% aqueous Toluidine blue (Rock, 1981) or with 0.5% Sudan IV in 800/0 ethanol (Foster, 1949).

In one experiment where seedlings were exposed to 12 h photoperiods of 0.5 or 1 klux or to darkness, light at both intensities inhibited primary-root elongation by 40% (Fig. 1). In two experiments where seedlings were exposed to 24 h photoperiods of 5 klux or to darkness, light inhibited primary-root elongation by 27 and 38 % (Fig. 2). In these latter two experiments light severely inhibited secondary-root formation (Fig. 3).

Fig. 1: Rate of primary-root elongation for seedlings grown in darkness (D) or in 12 h photo-periods of 0.5 or 1.0 klux of fluorescent light. Otherwise see Materials and Methods.

Fig. 2: Rate of primary-root elongation in two experiments (A and B) where seedlings were grown in darkness or in 24 h photoperiods of 5 klux.

When these cultures were transferred to darkness secondary-root production commenced and continued at a rate comparable to that of the dark controls. However, only 10 and 12% of these latter formed roots developed from primary-root areas which had previously been exposed to light.

Callus formation occurred from 66% of the seedlings exposed to 24 h photoperiods of 5 klux (Fig. 4). Only 16% of the dark controls developed similar calluses and these were far less extensive than those formed in the light.

Callus formation also occurred at the junction of the primary-root and the shoot in all of the above cultures. When seedlings with light-induced calluses were transferred to the dark, the calluses retained their unorganized appearance but in a few cases secondary-roots grew out of these calluses.

Secondary-roots formed in all of the light treatments developed chloroplasts but otherwise resembled those produced by the dark controls (Fig. 5). When dark controls were transferred to 24 h photoperiods of 5 klux, callus formation occurred along the primary- and secondary-root axes (Figs. 6, 7) and from secondary-root apices (Fig. 7) in 83 % of the cultures. These latter calluses eventually became organized into typical root apices which did not resemble nodules (Fig. 6). Callus formed along the root axes remained unorganized.

In both primary- and secondary-roots callus formation also occurred in both light and darkness, where lateral roots emerged (Figs. 6, 7). These calluses were more prominent in the light than in the dark.

Callus formation not associated with lateral root emergence, had a superficial origin in the subepidermal cortex. The phellogen of developing periderm was continuous with areas of callus proliferation which resembled lenticils. Callus cells were isodiametric and were loosely arranged in uniseriate rows. The callus producing meristem appeared to be a single cell layer external to cortical parenchyma cells. Callus cells stained positively for carbohydrates with Toluidine blue but they failed to stain positively for suberin with Sudan IV. Adjacent phellem cells stained positively with Sudan IV.

Light-induced callus which formed at different loci frequently united and completely encircled the root. Internally the callus originated from a single meristematic cell layer which was continuous around the root cortex. Following secondary growth of the vascular cambium, the callus producing meristematic layer was located in the secondary phloem. Cells produced by this lateral meristem were similar to those produced at the onset of callus formation and appeared to be the product of periclinal divisions in one direction. In no cases were lateral-root primordia or areas resembling them seen in association with these calluses or their meristems.

The development of callus formed by secondary-root apices was not examined.

With Bowenia serrulata (Webb, 1982 a) and 7. pumila (Webb, 1981, 1982 b) light induced secondary-root nodulation. Since auxin (Slankis, 1950, 1958) and cytokinin (Becking, 1975) can induce root nodulation in other gymnosperms or in angiosperms and since these two hormones may be involved in periderm development (Peterson, 1971), it is logical to hypothesize that light alters the hormonal balance in cycad roots which leads to nodulation with B. serrulata and 7. pumila, and to periderm-derived callus formation with D. edule. Callus formation by D. edule roots may be enhanced by the moist culture environment.

The observation that nodulation did not occur with D. edule in sterile culture suggests that other abiotic and/or biotic factor(s) must be required for nodule formation. Within the Zamiaceae there appear to be three patterns of nodulation. With Macrozamia rudely nodulation does not require a specific external stimulus (Lamont and Ryan, 1977). With B. serrulata (Webb, 1982 a) and Z. pumila (Webb, 1981,1982 b) light causes nodulation and with D. edule (an) unknown external factor(s) is required for nodulation.

Dioon edule seeds necessary for these experiments were donated by Mr. William Steen of The Cycad Society. This research was supported by a grant from the Office of Graduate Education and Investigation, University of Puerto Rico, Rio Piedras.