In vitro regeneration and secondary metabolites in cyrtanthus species.

Ph. D. University of KwaZulu-Natal, Pietermaritzburg, 2013.

Perennial geophytes form part of the diversified flora in southern Africa. The traditional medicinal system, integrates various plant components in the treatment of diverse ailments. In South Africa, bulbs are part of the extensively exploited floral resources for traditional medicine purposes. The reason for selective preference of certain groups of plant species or taxa, particularly geophytes, is rooted primarily on the beliefs that potent constituents are from the underground plant parts. The great surge of public interest in the use of plants as medicines however, assumes that plants will be available on a continuing basis. A vastly increasing human population coupled with the rapid degradation and fragmentation of natural habitats, exacerbate the threats posed by increasing demand on floral resources. The highly endemic members of the genus Cyrtanthus, most of which have limited geographic distribution ranges, are increasingly exploited for traditional medicines in South Africa. Bulbs of this species are the most preferred part for medicinal use, leading to the destructive harvesting of these plants. This form of plant harvesting poses threats to the long term sustainability of these plant resources in their natural habitats. Although sustainable harvesting of plant resources should be within the limits of their capacity for self-renewal, this seldom occurs owing to indiscriminate and destructive harvesting by commercial medicinal plant gatherers. As a consequence of this and other factors, intensive population decimation of a number of Cyrtanthus species is now evident and widespread, with some species threatened with extinction. The extinction of these species could lead to, in addition to the undesirable loss of genetic variability, loss of potential therapeutic agents. Conservation of these plant resources is therefore essential. The aim of this study was to establish efficient in vitro regeneration protocols for three threatened Cyrtanthus species (C. contractus, C. guthrieae and C. obliquus) endemic to southern Africa and explore the possible potential of improving the quality and quantity of bioactive secondary metabolites in culture. In vitro cultured twin-scale explants of the three selected Cyrtanthus species using different concentrations and combinations of 6-benzyladenine (BA) (0, 1.1, 4.4, 6.7, 8.9 μM) and naphthalene acetic acid (NAA) (0, 0.5, 1.1, 2.7 μM) in a 4 x 5 factorial treatment structure, established different optimal PGR combinations for shoot regeneration for each species. The highest shoot induction responses were obtained on MS medium with 4.4 μM BA/1.1 μM NAA for C. contractus and C. guthrieae and 6.7 μM BA/2.7 μM NAA for C. obliquus. The low concentration level of PGR requirements for shoot regeneration in C. contractus and C. guthrieae explants may suggest that the two species contain high enough endogenous hormones to induce shooting compared to those of C. obliquus. When the effect of different types and concentrations of cytokinins (CKs) [BA, kinetin (Kin), meta-topolins (mT), zeatin (ZT) and thidiazuron (TDZ)] on shoot multiplication were evaluated, 5 μM TDZ, 10 μM TDZ and 10 μM BA for C. guthrieae, C. contractus and C. obliquus respectively, were established as the optimum for shoot proliferation in each respective species. These results indicate, TDZ, a characteristically inexpensive CK, to be highly potent and effective in shoot proliferation of C. guthrieae and C. contractus. In terms of visual quality, shoots obtained from media supplemented with Kin and mT resulted in the best quality shoots in all three species at all concentrations tested. Furthermore, Kin also exhibited some auxin-like activitty by inducing rooting and callus on C. contractus and C. guthrieae cultures. The regenerated organogenic calli from C. guthrieae explants produced the optimum number of shoots through indirect organogenesis when transferred to MS medium supplemented with a combination of 0.1 μM picloram and 0.01 μM BA. An almost two-fold shoot proliferation frequency was obtained when the resulting callus-derived microshoots where subsequently transferred to the optimised shoot proliferation medium for the species. Regenerated shoots for all species were rooted successfully on half- and full-strength MS media without plant growth regulators, transferred to organic soil mix, and successfully acclimatised in greenhouse conditions. The developed micropropagation protocols provide a rapid and cost effective way of conservation, domestication and commercial cultivation of Cyrtanthus species. The levels of proline and polyphenolic compounds measured at intervals of three, four and five weeks from initial plantlet culture under different levels of salinity and osmotic regimes, increased in a stress-dependent pattern. The levels of these metabolites also showed a significant increase with an increase in the duration of plantlets under stress conditions. The highest proline concentration (9.98 μmol gˉ¹ FW) was recorded in C. contractus at 300 μM NaCl after five weeks. The high level of total polyphenolic compounds (147 mg GAE gˉ¹ DW) for the same species was however, recorded in the 150 μM NaCl stress treatment. The activity of proline dehydrogenase (PDH) (EC 1.5.99.8) was shown to decrease with an increase in proline levels from week three to week five in almost all stress conditions evaluated. The high levels, particularly of phenolic compounds obtained under osmotic and salinity stress conditions in this study present a promising potential for manipulating culture and/or growing conditions for improved secondary compound production and hence medicinal benefits. In a study of the growth dynamics and patterns of assimilate partitioning to primary and secondary metabolites in response to varying levels and combinations of C (carbon) and N (nitrogen) in the culture media of Cyrtanthus guthrieae, relative growth rate (RGR) increased proportionally with an increase in C concentrations up to 88 mM sucrose (0.58 dˉ¹) beyond which it was hardly influenced by further increases in C. In C-limited media regimes with growth saturating N conditions, alkaloid accumulation became favoured while polyphenol content increased with an increase in C levels in the medium, a characteristic pattern that appeared to be less influenced by the amount of N. Of the primary metabolites, only proteins showed small significant variations across different media treatments, with starch and soluble sugars increasing proportionately with C levels. From a medicinal perspective, with regard to polyphenolic compounds in C. guthrieae, growth media conditions that allow for high levels of C pools in the tissue would thus be favourable for the enhanced synthesis of this group of compounds. The medium conditions with 175 mM sucrose and 10.3 mM NH₄NO₃ gave the highest total polyphenol, flavonoid and proanthocyanidin levels with a moderate growth rate. Pharmacological evaluation of the monthly collected C. contractus bulbs indicated some impressive bioactivities particularly the cytotoxicity effects against human cancer cell lines and enzyme inhibition (AChE and COX) by the extracts collected in certain months of the year. Of notable interest were the cytotoxicity effects, AChE and COX enzymes inhibitory activity of the extracts collected in May and September. Similarly, some extracts from in vitro precursor-fed plantlet and callus cultures demonstrated some excellent bioactivity, against COX and AChE enzymes. The results obtained from this study also reflect on the involvement of the environment in the quality of the extracts produced on a month to month basis and further suggest the importance of coinciding collection and use of plant extracts with the best time of the year or month. The good AChE and COX enzyme inhibitory activity by some of these extracts is of significant importance in the treatment of Alzheimer’s disease and neuroinflammation. The extracts represents an important component of traditional medicine.