In vitro propagation of Dierama erectum.

Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Dierama is a genus of plants with a potential to be developed as ornamental plants. It falls under the Iridaceae family and comprises of 44 species. Dierama erectum Hilliard, an attractive species with horticultural potential is mainly found in rough wet grasslands. Its corms are used for enemas and treating stomach ailments in southern African traditional medicine. Due to its habitat transformation by afforestation and the exploitation of its underground parts (corms) in traditional medicine, this plant is among the most vulnerable and rare species within its genus. Seed parasitism by Urodon lilli also hampers its conventional propagation. The increase in demand for ornamental and medicinal plants increases pressure on wild plant populations. Micropropagation is a useful tool for clonal propagation of plants as it does not only help in alleviating pressure on wild plants but an effective micropropagation protocol could also provide a foundation for plant genetic transformation, which could result in the development and introduction of new ornamental varieties into commercial markets. This research was aimed at developing a micropropagation protocol for D. erectum to ensure readily available source material for medicinal and horticultural use as well as serving as an alternative for its conservation. Seed decontamination and germination were successful when 0.2% HgCl2 or 2.5% NaOCl + 1% Benlate® were used. However, for safety reasons, 2.5% NaOCl + 1% Benlate® was used in all subsequent experiments. The shoot regenerative capacity of leaf, hypocotyl and root explants obtained from in vitro germinated seedlings was evaluated by culturing them individually on MS medium supplemented with various concentrations of BA. Only hypocotyl explants produced adventitious shoots. Since no shoots or callus was produced from leaf and root explants, hypocotyl explants were used in the development of a micropropagation protocol. Different types and concentrations of cytokinins (BA, mT, KIN and Z) with or without NAA were evaluated for their effect on adventitious shoot production. Maximum shoot number per explant (4.20 ±0.51) was obtained in MS medium supplemented with 1.0 ìM Z after 8 weeks. This was followed by a combination of KIN (2.0 ìM) and NAA (0.5 ìM) resulting in a production of 3.67 ± 0.81 shoots per explant. For BA treatments, the highest shoot multiplication (3.20 ± 0.22 shoots per explant) was achieved when 2.0 ìM was combined with 1.0 ìM NAA. mT gave maximum shoot production (3.09 ± 0.99 shoots per explant) when 2.0 ìM mT was combined with 2.0 ìM NAA. The effects of photoperiod and light intensity were investigated for the purpose of optimizing shoot multiplication. An average of 12.73 ± 1.03 shoots per explant were obtained after 8 weeks from shoots grown in 16 h light at a 100 ìmol m-2 s-1 light intensity. The 24 h light treatments and a light intensity lower than 100 ìmol m-2 s-1 negatively affected growth and regeneration of D. erectum. These results highlighted the need for evaluating environmental conditions when developing micropropagation protocols. Corm induction experiments were done with the intention of facilitating acclimatization of D. erectum ex vitro. Various concentrations of ancymidol, activated charcoal and sucrose did not promote in vitro corm formation, thus auxins (IAA, IBA and NAA) were tested for their efficiency in rooting. Plants rooted successfully after 8 weeks on MS medium supplemented with 1.0 ìM IBA, yielded the longest roots (4.63 ± 0.70 cm) and an average root number of 2.73 ± 0.40. All NAA treatments resulted in stunted roots. Plants grown in vitro were potted in trays containing a 1:1 ratio of soil: vermiculite and placed in the mist house for 2 weeks. They were then transferred to the greenhouse for further acclimatization. After 2 months, plants had formed corms. The largest corms (0.45 ± 0.026 cm in diameter) were found in plants pre-treated with 0.5 ìM IBA. Maximum plant survival percentage (73%) was also associated with this treatment. A successful micropropagation system for Dierama erectum was therefore developed. The utilisation of this protocol can yield about 15137 plants from one explant in a year. This will expand our existing knowledge about micropropagation of plants in the genus Dierama and will be useful in the conservation of this species.