Thesis (MScAgric)--Stellenbosch University, 2017.ENGLISH SUMMARY: Extracts from the leaves of Stevia rebaudiana, a plant native to South America, have been
used as natural sweetener for centuries. With the global epidemic of obesity linked to
increased prevalence of diabetes, Stevia has attracted interest for use as a non-nutritive
sweetener (NNS). Unlike currently available NNS which are chemically synthesised (e.g.
sucralose), Stevia extracts represent naturally occurring NNS with no negative side effects
from its use. The sweet-to-taste compounds in Stevia are actually due to the accumulation of
secondary metabolites in the leaves, specifically two steviol glycosides (SGs, stevioside and
rebaudioside A). However, these SGs occur in low concentrations (between 2-4% of total
fresh weight) and show variability in plants grown by commercial scale agricultural
propagation. The plant also requires high irrigation inputs owing to its sensitivity to even
moderate water deficit.
Stevia is currently not a cash crop in South Africa (SA) but there is interest in establishing
commercial scale agricultural ventures to establish a Stevia economy. SA is also experiencing
a concerning rise in the number of new incidences of diabetes amongst its population and
recently approved the introduction of a sugar tax that is envisaged to reduce this excessive
sugar intake and over time improve the health and well-being of the population. The variable
SG yields and the high irrigation inputs required to produce them from the plant are
considered major restrictive factors toward establishment of a Stevia economy in SA - a
naturally water scarce country. Current propagation methods for Stevia are both laborious
and costly because the seeds are recalcitrant and plants have to be propagated via stem
cuttings or in vitro tissue culture.
Hairy root cultures have been widely used in plants of medicinal importance to obtain high
quantities of bioactive secondary metabolites, for use as pharmaceutical drugs.
Agrobacterium rhizogenes is utilised in this context to induce hairy root formation and a few
studies have investigated Stevia hairy root cultures but none have reported SG accumulation
in these cultures. This study attempted to create Stevia hairy root cultures expressing key
genes in the SG biosynthesis pathway and accumulating the two sweet SGs, stevioside and
rebaudioside A. Additionally, attempts were made to create Stevia hairy root cultures
overexpressing UGT74G1 and UGT76G1 (the two genes responsible for stevioside and
rebaudioside A accumulation respectively) with the intention of increasing SG production..
Although we demonstrated that A. rhizogenes could be transformed with the plant expression constructs and that this transformed A. rhizogenes could induce hairy roots from leaf
explants, tandem mass spectrometry analyses of root extracts did not identify either
stevioside or rebaudioside A. We suspect that the lack of photosynthetic capacity in hairy
root cultures resulted in the unavailability of key intermediate substrates for SG biosynthesis
that have been proposed to be produced during photosynthesis. However, we are currently
investigating if these hairy root cultures could be primed for SG accumulation by growing
them in the presence of the proposed intermediate substrates which are available
commercially at low cost.AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar nie
