In vitro studies and phytocompound analysis in Lessertia frutescens (Fabaceae)
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Date
2011
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Abstract
The cancer bush (Lessertia frutescens L.) is an important leguminous perennial native to
southern Africa and has been used for centuries in traditional medicine by the continent’s diverse
cultural groups. Like many other legumes, the seeds of this species exhibit dormancy. Moreover,
woody plants are typically difficult to propagate in in vitro culture systems. But in vitro shoot
cultures are valuable in providing an alternative means of deriving desired secondary metabolites
or phytocompounds, under controlled conditions. This study describes novel protocols for
breaking seed dormancy, rapid and efficient in vitro propagation, bioreactor culture, and
comprehensive phytochemical data following screening and analysis of in vitro and field extracts
of L. frutescens. Experiments using physical, mechanical and chemical pre-sowing treatments
were conducted to determine the germination response of this species. The results indicated that
seeds of L. frutescens exhibited exogenous dormancy due to the inhibitory effect of the hard coat
on germination. Seed dormancy was released by mechanical scarification in which 100 %
germination was achieved. In vitro propagation studies using single node explants in Murashige
and Skoog (MS) medium supplemented with combinations of different concentrations of
benzyladenine and naphthaleneacetic acid revealed a maximum number of 10 shoots per explant
in solid medium, and 12.9 shoots per explant in liquid medium inside a temporary immersion
bioreactor. Indirect shoot organogenesis and plant regeneration using rachis and stem segments
was achieved with the highest percentage of explants forming shoots (88.8 %) from rachis
explants cultured onto MS medium supplemented with thidiazuron. Direct shoot organogenesis
from hypocotyl and cotyledon segments was also achieved in L. frutescens. The highest shoot
regeneration using hypocotyls (83 %) was obtained in MS medium supplemented with kinetin
whilst the highest shoot regeneration using cotyledons (46 %) was obtained in MS medium
supplemented with kinetin in combination with benzyladenine. Successful rooting (up to 80 %)
and acclimatization (up to 90 % survival rate) was attained. Spectrophotometric and gravimetric
methods indicated that saponins were the most abundant, followed by phenolics, flavonoids and
then alkaloids in in vitro leaf extracts then in field leaf extracts and seed extracts, respectively.
After qualitative analysis these extracts were also found to contain tannins, phlobatannins and
cardiac glycosides of medicinal interest. By using gas and liquid chromatography the presence of
the medicinally important L-canavanine, gamma amino-butyric acid and D-pinitol was verified
in in vitro leaf, field leaf and seed extracts. In vitro leaves had higher quantities of all
compounds, except for D-pinitol. Phytocompound analysis of shoots derived from several of the
cytokinin-enhanced media showed that these organs contained higher quantities of L-canavanine
compared to the control. This study, therefore, highlights the potential techno-economic
production of medicinal phytocompounds from in vitro leaves of L. frutescens following large
scale production using the protocols described in this study.
Description
Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.
Keywords
Medicinal plants--Southern Africa., Legumes., Theses--Botany.