Development of a hairy root bioreactor from Stevia rebaudiana to produce steviol glycosides

Mthembu, Zanele (2017-12)

Thesis (MScAgric)--Stellenbosch University, 2017.

Thesis

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.

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