Effects of over-expressing the AgGPPS2 gene in Salvia stenophylla (Burch. ex Benth) on terpenoid biosynthesis

Musarurwa, Hannibal Tafadzwa (2013-03)

Thesis (PhD)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Plant secondary compounds have been exploited as medicines, flavourants, incense and are widely used for different culinary purposes. Efficacy of herbal remedies is determined by the phytochemical profile which is dependent on the species, locality and growth conditions. Salvia stenophylla (Burch. ex Benth.) is a local aromatic sage growing in almost all South African biomes, with the volatile compound accumulation varying depending on season, locality and genotype. Salvia stenophylla extracts have proven anti-inflammatory, anti-plasmodial, anti-oxidant and anti-anxiety properties due to the presence of essential oils, flavonoids and caffeic acid derived phytochemicals. As a result, S. stenophylla extracts are increasingly being used for the formulation of pharmaceutical and cosmeceutical products. However, these industries largely depend on the wild populations for raw materials, and increased commercialization of sage-based natural products and medicine exerts harvesting pressure. This might reduce the sustainability of herbal medicines since there is no formal cultivation of S. stenophylla in South Africa. Medicinal plant biotechnology provides options for propagation and manipulation of herbal plant to increase synthesis of secondary compounds. The aims of this study were to develop tissue culture propagation system so as to provide an alternative to wild-harvesting, to improve essential oil quality and accumulation in S. stenophylla by applying third generation biotechnological tools to alter terpene biosynthesis via heterologous gene expression aided by Agrobacterium-gene transfer and to charaterize the resultant metabolite profiles using thin layer chromatography, gas chromatography mass spectrometry and liquid chromatography mass spectrometry. Initially, seeds were germinated in-vitro on one-tenth Murashige and Skoog medium (MS) (1962). Seeds were decontaminated by washing them in 3.5% (w/v) hypochloride and were either scarified using 70% (v/v) sulphuric acid, placed on media with smoke solution or both. Both scarification and smoke supplementation induced germination, but seeds subjected to both treatments, as a combination, showed very poor germination. Seedlings produced were used to establish an efficient tissue culture system for S. stenophylla. Shoot tips, nodal and basal explants were placed on MS media with different plant growth regulator (PGR) combinations and concentrations. The best plantlet regeneration and shoot elongation were observed on plants on medium supplemented with 5.7 μM indole acetic acid (IAA) and 8.9 μM N-6-benzyladenine (BA), producing 4-6 shoots per explant with 6.67 cm mean length. Treatment with 4.5 μM 2,4 dichlorophenoxyacetic acid (2,4 D) did not produce shoots but had the highest biomass production, which was all in the form of callus. The resultant shoots from the micropropation system had similar metabolites as wild type plant and also they had some compounds that were not detected in their wild type counterparts. The effects of nitrogen, potassium, water stress, and phytohormones on metabolite accumulation were also studied in a S. stenophylla microplant system. Plant growth regulator free half strength MS medium encouraged the most prolific growth. Increasing potassium and nitrogen concentrations correlated to the accumulation of (-)-α-bisabolol whilst changes in other major compounds like ß-bisabolene, α-muurolene, α-patchoulene, and D-limonene were insignificant. To investigate the effects of water stress on metabolite accumulation and profile, water stress was induced using sorbitol and polyethyl glycol. Reduced water availability only negatively affected rooting in-vitro whilst the chemical profile was not affected. The study also focused on terpene production in S. stenophylla by over-expressing the heterologous geranyl diphosphate synthase gene (AgGPPS2) from Abies grandis, using Agrobacterium tumefaciens (EHA105). The AgGPPS2 gene was ligated to a pCAMBIA1301 vector which was cloned in EHA105 and this in turn was used to transform S. stenophylla. Resultant transgenic plantlets exhibited normal growth characteristics but showed variation in the metabolite accumulation. GC-MS analysis showed a 6% increase in (-)-α-bisabolol accumulation whilst 3-δ-carene, α-pinene and camphor were lowered. Solvent extracts were analysed using LC-MS and these had rosmarinic acid in greater abundance in transgenic than in the wild type plants. However, the chemical profiles of the wild type and transgenic plants showed some similarities suggesting AgGPPS2 expression only alters the abundance of some secondary compounds, whilst the overall integrity of the metabolome is maintained. Unlike most metabolite engineering studies elsewhere, this is the first attempt a local South African sage has been genetically engineered to enhance terpene biosynthesis. This is crucial in an economy that is increasing becoming plant based for pharmaceutical, industrial and food needs. Further studies are required to elucidate the efficacy of transgenic and in-vitro plant-derived extracts.

AFRIKAANSE OPSOMMING: Plant sekondêre produkte word gebruik as medisinale-, geur-en reukmiddels en word ook as algemene bestandeel gebruik in kookkuns. Die doeltreffendheid van kruie middels word bepaal deur die fitochemiese profiel. Hierdie profiel is afhanklik van die spesie, ligging en groei kondisies. Salvia stenophylla (Burch. ex Benth.) is ‘n plaaslike aromatiese salie wat voorkom in byna al die biome van Suid-Afrika , waar die versameling van vlugtige verbindings varieer op grond van die seisoen, ligging en genotipe. Salvia stenophylla ekstraksies is bewys om anti-inflamatories, antiplasmodiaal, anti-oksidant en anti-angs eienskappe te besit as gevolg van die noodsaaklike olies, flavonoïede en afgeleide kaffieksuur fitochemikalieë wat die plant besit. As gevolg word S. stenophylla ekstraksies meer en meer aangewend in die ontwikkeling van farmaseutiese en kosmetiese produkte. Hierdie industrieë benodig rou plant material vanuit die natuurlike populasies wat druk op die biome sit as gevolg van die verhoogde kommersialisering van salie-gebaseerde natuurlike produkte en medisyne. Gevolglik is die volhoubaarheid van medisyne afkomstig vanaf kruie soos salie in bedwang omdat geen formele kultivasie van S. stenophylla in Suid-Afrika al in plek gestel is nie. Medisinale plant biotegnologie bied moontlikhede vir kultiveering, voortplanting en manipuleering van kruie om sodoende die produksie van sekondêre verbindings in kruie te verhoog. Die doel van hierdie studie was om ‘n weefselkultuur voortplantingssisteem te ontwikkel om sodoende ‘n alternatief te bied vir wild oesting. Die studie het gefokus op die verbetering van noodsaaklike olie kwaliteit en akkumulasie in S. stenophylla deur die gebruik van derde generasie biotegnologiese gereedskap om terpeen biosintese te modifiseer. Hierdie is bereik deur weefselkultuur en heterologiese geen uitdrukking wat aangehelp is deur Agrobacterium geen oordrag. Die gevolglike metaboliet profiele was gekarakteriseer deur gebruik te maak van dun laag chromatogragie, gas chromatografie massa spektrometrie en vloeistof chromatografie massa spektrometrie. Aanvanklik is die sade ontkiem in-vitro op een-tiende Murashige en Skoog medium (MS) (1962). Dekontaminasie van die sade is gedoen deur die sade te was in 3.5% (w/v) hipochloried waarna die sade óf geskarifideer is deur te was met 70% (v/v) swawelsuur óf op medium geplaas is wat ‘n rook oplossing bevat óf albei. Beide skarifikasie en rook aanvulling het ontkieming aangehelp, maar sade wat aan albei behandelings blootgestel is, het baie swak ontkieming getoon. Saailinge was gebruik om ‘n effektiewe weefselkultuursisteem vir S. stenophylla te produseer. Stingel punte, nodale en basale eksplante was op MS medium geplaas met verskillende kombinasies en konsentrasies van verskeie plant groei reguleerders (PGR). Die beste plant regenerasie en stingel verlenging was geobserveer met plante op medium aangevul met 5.7 μM indool asynsuur (IAS) en 8.9 μM N-6-bensieladenien (BA), waar dit 4 tot 6 stingels per plant met ‘n gemiddelde lengte van 6.67 cm gegroei het. Behandeling met 2,4 Dichlorophenoksiasynsuur (2,4 D) het nie stingels produseer nie, maar het die hoogste biomassa produksie getoon in die vorm van kallus. Die gevolglike stingels van die mikrovoortplantingsisteem het soortgelyke metaboliete as die wilde tipe plante, maar verbindings wat nie in die wilde tipe plante voorkom nie was ook ontdek. Die effek van stikstof, kalium, water stres en fitohormone op metaboliet akkumulasie was ook bestudeer in S. stenophylla mikroplantsisteem. Plant groei reguleerder vrye half sterkte MS medium het die produktiefste groei aangemoedig. Verhoging van kalium en stikstof konsentrasies het gekorreleer met die versameling van (-)-α-bisabolol en terwyl veranderinge in ander belangrike verbindings soos ß-bisaboleen, α-muuroleen, α -patchouleen en D-limoneen onbeduidend was. Om die effek van water stres op metaboliet- akkumulasie en -profiel te ondersoek was water stres geinduseer deur sorbitol en poli-etielglikol te gebruik. Die verlaging van water beskikbaarheid het slegs ‘n negatiewe invloed op in-vitro wortelvorming gehad terwyl die chemiese profiel onveranderd gebly het. Die studie het ook gefokus op die terpeen produksie in S. stenophylla deur die ooruitdrukking van die heterologiese geranieldifosfaatsintase geen (AgGPPS2) vanaf Abies grandis met behulp van Agrobacterium tumefaciens (EHA105). Die AgGPPS2 geen was geligeer in ‘n pCAMVIA1301 vektor wat geklooneer was in EHA105 en dit op sy beurt was getransformeer in S. stenophylla. Die gevolglike transgeniese plante het normale groei ten toongestel, maar het variase in metaboliet akkumulasie getoon. GC-MS analises het gewys dat ‘n 6% toename in (-)-α-bisabolol versameling terwyl 3-δ-kareen, α-pineen en kamfor verlaag het. Opgelosde ekstraksies was geanaliseer deur VC-MS en het gewys dat roosmariensuur in groter hoeveelhede in transgeniese plante as wilde tipe plante akkumuleer. Nogtans het die chemiese profiele van die twee genotipes sommige ooreenkomste getoon, wat voorstel dat AgGPPS2 uitdrukking slegs sekere sekondêre verbindings menigte male vermeerder terwyl die algehele integriteit van die metaboloom gehoue bly. In teenstelling met die meederheid studies oor metaboliet ingenieuring wat elders plaasvind, is hierdie die eerste poging om ‘n plaalike Suid-Afrikaanse salie genieties te ingenieur met die oog om die terpeen biosintese te verbeter. Hiedie is krities in ‘n ekonomie wat voortdurend vorder om plant gebaseerde produkte te maak vir farmaseuties, industrieël en voedsel behoeftes. Verdere studies is nodig om helderheid te gee oor die effektiewiteit van transgeniese en in-vitro plant afkomstige ekstraksies.

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