Thesis submitted in fulfilment of the requirements for the degree
Master of Technology: Horticultural Sciences
in the Faculty of Applied Sciences at the
CAPE PENINSULA UNIVERSITY OF TECHNOLOGY
Supervisor: Dr L Kambizi
Co-supervisor: Dr NP Makunga
Cape Town
December 2013 / Agathosma betulina (Berg.) Pillans, previously known as Barosma betulina, is a
member of the Rutaceae family, and indigenous to the fynbos botanical biome of the
Western Cape of South Africa. It is commonly known as buchu. Extracts as well as
powdered leaves have traditionally been used for the treatment of various ailments.
The increase in the international demand for A. betulina for health as well as food
and beverage benefits, have raised concerns over exploitation of wild populations
and the lack of horticultural information necessitates this study to evaluate the
propagation of this economical important species. The main objective of this study
was to establish a simple and highly productive micropropagation protocol for A.
betulina through experimenting with nodal explants.
Testing of the effect of various treatments (physical scarification, chemical
scarification, GA, stratification, smoke and combinations thereof) on the in vitro
germination of A. betulina seeds was done to elucidate the factors which control seed
germination. The study revealed that the physical scarification and smoke-induced
germination had a significant effect on germination percentages. In terms of
germination rate, the radical generally started to appear after approximately 10 days
in the physical scarification with smoke treatment.
Initial decontamination methods with the exposure of various concentrations of
NaOCl gave fatal results, however 1.5% NaOCl had more phenolic reactions rather
than fungal or bacterial contamination. Interestingly, contamination rates of
explants were influenced by the stage of maturity of the explant material. This plant
material was used to test different strengths of regeneration media, to ensure that the
explants receive ample nutrients. Results made exhibited that ½ MS was the best
strength for growing A. betulina nodal explants. Compared comparison between in
vitro derived explants and ex vitro collected explants showed that the ex vitro derived
explants had significant results, but the explants lost vigour soon after the initial
exponential growth leading to the explants dying off. Furthermore, ex vitro
decontaminated plant material was not economically viable to continue with.
Seedlings derived from germinated seeds appeared to be the preferred method of
propagation as this spent the least time in culture and produced a stable plant with
an established root system, which is essential during the hardening off process after
in vitro growth. When exposing nodal explants to phytohormone 2,4-D it responds
best to dosages 0.5mg Lˉ¹ and 1mg Lˉ¹. Phytohormone BA was very effective in
producing soft friable callus. The best results were shown when 0.5mg Lˉ¹ BA was
applied to ½ MS media. For both shoot length and multiple shoot production, a
combination of phytohormones BA-NAA (1: 0.5mgLˉ¹) had the most significant
results. Interestingly, a higher phytohormone concentration of NAA is necessary to
develop multiple adventitious roots. The effect of 3mg Lˉ¹ was significant in that it
resulted in multiple adventitious roots, but fewer calli was observed in this treatment.
Micropropagation becomes valuable as little attention between subcultures is
needed; making it less labour intensive compared to conventional nursery
propagation systems where weeding watering and spraying of plants are labour
intensive.
In the traditional world of medicine, more so in Southern Africa, extracts are prepared
by adding boiling water to the plant material; however commercial ethanol is used as
an extractant. Establishment of the essential oil quality of the in vitro cultures post
exposure to various treatments was done. Analysis of essential oils from A. betulina
resulted in the identification of twenty one compounds. The results showed
qualitative as well as quantitative differences amongst the samples used in the study.
The highest relative concentration of limonene was observed in the callus of nodal
explants after it was exposed to 0.5mg lˉ¹ NAA. No pulegone was found in this
treatment making it ideal for limonene production. This suggests that liquid culture
with the same treatment may produce more calli making it ideal for the production of
limonene.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:cput/oai:localhost:20.500.11838/841 |
| Date | January 2013 |
| Creators | Witbooi, Hildegard |
| Contributors | Kambizi, L., Dr, Makunga, N.P., Dr |
| Publisher | Cape Peninsula University of Technology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | http://creativecommons.org/licenses/by-nc-sa/3.0/za/ |
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