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. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1988.

Les outils de la biologie moléculaire, notamment l'utilisation des méthodes de séquençage de 2e et 3e génération ont eu un impact énorme sur la biologie. Leur application à la recherche sur les végétaux a cependant pris du retard, et les bases de données ne sont donc pas complètes. Il manque notamment de données pour une famille de gymnospermes, les cycadales. Zamia stevensonii est une cycade endémique du Panama récemment décrite. Comme tous les représentants de ce clade, elle forme une symbiose avec des cyanobactéries fixatrices d'azote dans des racines spécialisées, les racines coralloïdes. Ce projet a comme objectif d'abord de produire un transcriptome de référence, le plus complet possible, de Zamia stevensonii. Celui-ci sera ensuite utilisé pour identifier des orthologues partagés entre les espèces hôtes de cyanobactéries, un premier pas pour cibler des gènes impliqués dans ce type de symbiose toujours méconnu. Le transcriptome produit ici, à partir de 11 échantillons différents, provenant de 4 tissus différents, chacun à un stade de développement différent, est le résultat d'un séquençage Illumina MiSeq. Les données ainsi obtenues sont de bonne qualité et complètes, une grande proportion des contigs assemblés a des ORFs reconnaissables et les scores BUSCO sur trois lignées suggèrent un échantillonnage complet. La recherche d'orthologie entre les espèces hôtes de cyanobactéries n'a cependant relevé aucun orthologue partagé spécifiquement par ces espèces. Ce résultat suggère qu'au moins une espèce hôte de cyanobactéries n'utilise pas les mêmes processus moléculaires pour le maintien de la symbiose que les autres. La poursuite de la recherche dans le domaine pourrait considérer toutes les parties prenantes, l'holobionte, par des méthodes de transcriptomique, pour identifier les processus moléculaires permettant la symbiose avec les cyanobactéries. / Molecular biology techniques, and especially the use of 2nd and 3rd generation sequencing technologies, have revolutionized biological research. However, their use for plant biology research has lagged behind, and the current databases are left with important gaps. Of the data available, very little is known for a particular group of gymnosperms, the cycads. One such cycad, Zamia stevensonii, has recently been described in Panama. As all other cycads, it produces specialised secondary roots, coralloid roots, to host nitrogen-fixing symbiotic cyanobacteria. This project has as main objective to produce a reference transcriptome, as complete as possible, for Z. stevensonii. The produced assembly will be used to search for orthologous sequences with all other known hosts of nitrogen-fixing cyanobacteria. The sequences identified will be an important first step towards a better understanding of this lesser-known symbiosis. The transcriptome produced here contains sequences obtained from 11 tissue samples, from two sibling plants, including 4 different organs at different stages of development, produced by sequencing on the MiSeq Illumina platform. The data obtained is of good quality and complete, most filtered contigs have readable ORFs and BUSCO scores indicate sufficient sampling. Interestingly, the ortholog research for all nitrogen-fixing cyanobacteria hosts revealed no shared sequence between all host plants that would be unique to this specific symbiosis. This suggests that at least one host plant uses a different pathway to maintain the symbiosis. Future experiments could consider metatranscriptomics for the study of this type of nitrogen-fixing symbiosis and the molecular pathways involved.