An investigation into in vitro culture and phytochemical aspects of some members of the order Cycadales.
The present-day cycads represent the diverse, modified remnants of a much larger group of gymnosperms which flourished in the Mesozoic Era. The approximately 148 surviving species of the Cycadales are sparsely distributed through tropical and sub-tropical floras in a variety of habitats. About one-half of the extant taxa are considered endangered, vulnerable or rare and, because of their scarcity and decorative appeal, have attracted much public interest. Their slow growth rate, the paucity of viable seeds and limited potential for vegetative reproduction severely limit both the natural regeneration and the controlled propagation of cycads. Over the past 40 years, various attempts have been made to establish in v~o systems for cycad culture but none has been successful in establishing a functional protocol for the artificial propagation of these plants. The author has made renewed attempts to establish in vitro cultures from a range of haploid and diploid tissues from South African Encephalartos and Stangeria species. Callus proliferation was readily obtained from most explant sources of most species using a variety of media. Addition of the growth factors 2,4-dichlorophenoxyacetic acid and kinetin in the 10[-7] to 10[-6]M range was beneficial but not essential. Culture vessels which allowed relatively free gaseous interchange were advantageous and dark conditions were marginally better than constant light. Explants from cycad taxa which are mesic in habit gave a more rapid response than similar explants from xeric plants. Attempts to induce any form of differentiation other than, or after, callus formation were unsuccessful in all Encephalartos cultures, but two forms of morphogenesis were obtained from Stangeria-derived material. Megagametophytic tissue occasionally developed spherical outgrowths analogous to coralloid root primordia. More significantly, primary root cultures after callus formation, subculture and transfer to a light environment, regularly gave rise to meristematic zones and subsequent leaf emergence. This is the first recorded case of in vitro morphogenesis of a South African cycad. The order Cycadales shows several distinctive phytochemical features, principally the presence of the unique methylazoxymethanol glycoside toxins and a-amino-s-methylaminopropionic acid together with some unusual phenolic compounds, flavanoids, carotenoids and cyclitols. Stangeria differs from other cycads in at least two phytochemical aspects; the absence of biflavonoids in the leaves and the absence of rhamnose and methyl rhamnose in the hydrolysed mucilages. These diffe.rences may indicate broader physiological differences which would in turn explain the observed differences in morphogenetic competence of tissues from Stangeria and Encephalartos. Analyses of various tissues from these and other cycad taxa were performed with respect to moisture, protein, enzyme, toxin and hydrocarbon content. Significant differences, both between organs and between taxa, were noted. The results of peroxidase analyses were particularly important in that high levels of this enzyme correlate with the rapidity of callus formation in vitro. Furthermore, a sharp increase in peroxidase activity signals the onset of callogenesis in Stangeria megagametophyte cultures. An important incidental aspect of the phytochemical analyses is that of potential value of these data to the taxonomist. In particular, the leaf wax hydrocarbon profiles appear to be species-specific and are ideally suited to processing by numerical taxonomy computer programmes. It is anticipated that extension of this work will make a significant contribution to the resolution of existing problems in cycad taxonomy and, additionally, provide a means to construct phylogenetic sequences in the order.