Generation of clonal microplants and hairy root cultures of the aromatic medicinal plant Salvia runcinata L.f.

Figlan, Sandiswa (2012-12)

Thesis (MSc)--Stellenbosch University, 2012.


ENGLISH ABSTRACT: Bacterial and fungal pathogens have developed numerous defence mechanisms against antimicrobial chemical agents, and resistance to old and new produced drugs are on the rise. Discovery of natural products derived from plants with diverse chemical structures and novel mechanisms of action to treat these notorious pathogens is a priority. Biotechnology (discussed in Chapter 1) has much to offer as a pharmacological tool and in the general study of medicinal plants. The Genus Salvia (Lamiaceae) has gathered much interest as these plants manufacture a diverse range of secondary metabolites including flavonoids, tannins and terpenoids. Of particular interest are the terpenoids which are largely implicated in the efficacy of Salvia plants as traditional medicines contributing to their pharmacological actions (discussed in Chapter 2). Due to the importance of these plants as herbal remedies, in this study, biotechnological techniques such as tissue culture and Agrobacterium-mediated transformation were applied on Salvia runcinata L.f., a South African medicinal plant, in an attempt to enhance the metabolomic profile and its bioactivity. Like so many other sages, S. runcinata has been used in folk medicine to treat a variety of ailments. Application of biotechnology was viewed as an important value adding platform for this species, assisting with its commercialisation for the cosmeceutical and pharmaceutical industries. Therefore the study had three foci: (1) to determine the seed germination behaviour and optimal conditions for micropropagation; (2) to develop a protocol that would be efficient whilst being simple for genetic transformation; and lastly, (3) to conduct phytochemical studies on in vitro generated S. runcinata transgenic hairy root and in vitro organ cultures by comparing these to glasshouse plants as potential therapeutic sources of natural compounds used in the treatment of infections in plants and humans. Data generated is thus summarised in three research chapters and Chapter 3 describes the formulated procedures assisting with in vitro seed germination and micropropagation of S. runcinata. The efficacy of smoke and scarification treatments for germination improvement was initially tested coupled to the evaluation of different hormonal combinations and different explant types which would aid with inducing adventitious shoot formation in vitro. The most effective germination treatment proved to be a 3 min exposure of seeds to 25% (w/v) H2SO4 combined with a concentration of 10-5 M smoke solution, resulting to more than 80% germination. Shoot proliferation was significantly higher using nodal explants with the addition of 4.43 μM BA. The protocol established in this part of the study is viable for large scale commercial production of S. runcinata as it would yield 1296 to 46656 viable plants in 4 to 6 months from one nodal explant. Micropropagation was applied also as a pre-emptive measure to ease pressure on the wild plants as the demand for S. runcinata is anticipated to increase due to its growing economic value as it is one of two South African sages with epi-α-bisabolol that is sought after by the pharmaceutical and cosmeceutical industries. This makes the protocol developed in this part of the study suitable for ex situ conservation of S. runcinata plantlets. Evaluations on the transgene transfer capacities of two different agropine strains (A4T and LBA 9402) of Agrobacterium rhizogenes to induce hairy root cultures of S. runcinata explants on nodal and leaf explants were conducted (reported in Chapter 4). Hairy roots formed 3 to 4 weeks after inoculation of the explants and these agropine strains showed different abilities for genetic transformation with the LBA 9402 strain producing significantly more roots on each explant compared to the A4T strain (P=0.0075). However, none of the LBA 9402 derived clones and only 2 clones generated through A4T transformation survived subculturing. The polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence and transcription (respectively) of rol A, rol B, rol C and ags genes which are mobilised from the transfer-DNA (T-DNA) fragment of the root-inducing (Ri) plasmid of A. rhizogenes to the plant genome during transformation. The two A4T clones, termed here A4T3 and A4T5, were stably transformed, Southern blot analysis using rol A as a probe further validated the integration of one copy of the rol A gene. Transformed hairy roots, untransformed roots from tissue cultured plants, tissue culture-derived plants and glasshouse-grown plants were profiled for secondary metabolites by thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) in Chapter 5. In this part of the study, it is clear that the use of tissue culture as a propagation system did not negatively affect the volatile compound profile of S. runcinata and plants had a similar essential oil content to that reported by Kamatou et al. (2008), leading to a conclusion that in vitro plants maintained their biochemical integrity even under an alternative micro-controlled environment. Similarly to others, Ri-transformation was explored as an avenue to alter secondary metabolism creating inter-clonal variation. Transformed clones were distinguishable, displaying more of some primary metabolites including sucrose, galactose, sorbose and fructose than the leaf extracts. With the current GC-MS methods used, this clear distinction was not obvious at the secondary metabolite level. In general, solvent extracts (acetone and methanol:dichloromethane (MetOH: DCM) (1:1 v/v) exhibited good to moderate antibacterial activity with the minimum inhibitory concentration (MIC) values ranging from 0.39 to 0.78 mg ml-1. However, in vitro plant cultures were the most potent against two Gram-negative bacterial strains: Escherichia coli (ATCC 11775) and Klebsiella pneumoniae (ATCC 13883), and two Gram-positive bacterial strains: Bacillus subtilis (ATCC 6051) and Staphylococcus aureus (ATCC 12600). The hairy root extracts did not show any activity against fungi, Fusarium subglutinans (MRC 0115) and Fusarium proliferatum (MRC 6908). Micropropagation therefore proves to be an interesting avenue for commercial production of S. runcinata, supplying plants with an improved pharmacological activity. Hence the biotechnological approach applied here is a viable strategy for the production of medicinal bioactives from S. runcinata.

AFRIKAANSE OPSOMMING: Bakterieë en fungi patogene het baie verskeie meganismes ontwikkel teen antimikrobiese chemiese agente, en weerstand teen ou en nuwe chemise stowwe is besig om te vergroot. Daarom is dit belangrik om natuurlike plantaardige produkte met diverse chemiese strukture en unieke werkings meganismes te ontdek waarmee hierdie berugte patogene beveg kan word. Biotegnologie (wat in Hoofstuk 1 bespreek word) kan gebruik word as 'n farmakologiese hulpmiddel in die algemene studie van plante. Die Klas (Genus) Salvia (Lamiaceae) het al baie aandag getrek aangesien hierdie plante 'n wye reeks sekondêre metaboliete vervaardig wat flavonoïede, tanniene en terpenoïede insluit. Veral van belang is die terpenoïde wat betrokke is by die doeltreffendheid van die Salvia plante as tradisionele medisyne, aangesien dit bydra tot hulle farmalogiese aksie (wat in Hoofstuk 2 bespreek word). Aangesien hierdie plante sulke belangrike kruie is, word daar in hierdie studie, biotegnologiese tegnieke soos die kweek van weefsel en Agrobacterium-bemiddelde transformasie op Salvia runcinata L.f. toegepas om die metabologiese profiel en die bioaktiwiteit daarvan te verbeter. Soos baie van die salies is S. runcinata tradisioneel dikwels gebruik om allerhande siektetoestande te behandel. Die toepassing van biotegnologie word beskou as 'n belangrike manier om waarde by te voeg sodat hierdie plant kommersieei deur die kosmetiese en farmakeutiese bedrywe gebruik kan word. Daarom is daar op drie dinge gefokus: (1) die ontkiemings gedrag van saad en die optimale toestande vir mikrovoortplanting (2) die ontwikkeling van protokol wat eenvoudig maar doeltreffend is vir genetiese transformasie, en die (3) fito-chemise studies op in vitro genereerde S. runcinata transgeniese harige wortels en in vitro orgaan kwekings deur om hulle te vergelyk met kweekhuis plante as potentiële terapeutiese bronne van natuurlike samestellings vir die behandeling van infeksies in beide plante en mense. Die data wat gegenereer is, is opgesom in drie hoofstukke, en in Hoofstuk 3 word die prosedures wat gebruik word in die in vitro saad ontkieming en die mikro voortplanting van S. runcinata, bespreek. Die doeltreffendheid van rook en skarifikasie behandeling vir die verbetering van ontkieming is eers getoets en gekoppel aan die evaluering van verskillende hormoonkombinasies en verskillende eksplant tipes wat lei tot die formasie van uitloopsels in vitro. Daar is gevind dat die effektiefste behandeling vir ontkieming, 'n 3-minuut blootstelling van saad aan 25% (w/v) H2SO4 gekombineer met 'n konsentrasie 10-5 M rook oplossing is. Dit het gelei tot meer as 80% ontkieming. Daar was baie meer uitloopsels toe nodale eksplante gebruik is met die byvoeging van 4.43 μM BA. Die proktokol wat hier gevestig is, kan op groot skaal gebruik word vir die kommersiële produksie van S. runcinata, want 1296 tot 46656 lewensvatbare plante kan binne 4 ot 6 maande van een nodale eksplant gemaak word. Mikro voortplanting is toegepas as 'n voorkomende maatreel om die druk op die natuur te verminder omdat daar verwag word dat die vraag na S. runcinata sal toeneem na gelang die groeiende ekonomiese waarde daarvan toeneem. Dit is een van twee Suid-Afrikaanse salies met epi-α-bisabolol wat deur die farmakeutiese en die kosmetiese bedrywe gebruik word. Dit beteken dat die protokol wat hier ontwikkel is, geskik is vir die ex situ bewaring van S. runcinata plante. Die transgeen oordrag van twee verskillende agropien tipes (A4T and LBA 9402) van Agrobacterium rhizogenes is geevalueer (en in Hoofstuk 4 beskryf). Harige wortels het 3 tot 4 weke na die inenting van die eksplante gevorm en hierdie agropien tipes het verskillende vermoëns vir genetiese transformasie getoon, met die LBA 9402 tipe wat baie meer wortels op elke eksplant voorgebring het in vergelyking met die A4T tipe (P=0.03116). Geen van die LBA 9402-afgeleide klone en slegs 2 klone wat deur A4T transformasie genereer is, het oorleef. The polimerase ketting reaksie (PCR) en die teenoorgestelde trenskriptasie-polimerase (RT-PCR) ketting reaksie het die teenwoordigheid en transkipsie (onderskeidelik) van rol A, rol B en rol C en ags gene, wat oorgedra word deur die oordrag DNA (T-DNA) fragment van die wortel induserende (Ri) plasmied van A. rhizogenes na die plant genoom tydens transformasie, bevorder. A4T klone, hier A4T3 and A4T5 genoem, is stabiel transformeer. Southern blot ontleding het met die gebruik van rol A, die integrasie van een kopie van die rol A geen, bevestig. In Hoofstuk 5 is transformeerde harige wortels, ongetransformeerde wortels van weefsel gekweekte plante, weefsel gekweekte plante, en kweekhuis plante deur dun-laag chromatografie (TLC) en gas-chromatografie-massa spektrometrie (GC-MS) geprofiel vir sekondêre metaboliete. In hierdie deel van die studie is dit duidelik dat die gebruik van weefsel kwekery as 'n voortplantsisteem nie 'n negatiewe effek gehad het op die vlugtige samestelling profiel van S. runcinata nie en dat plante 'n sootgelyke essentiële olie inhoud het as wat deur Kamatou et al. (2008) bevind is. Dit lei tot die gevolgtrekking dat in vitro plante hulle biochemiese integriteit behou selfs onder alternatiewe mikro-beheerde omgewings. Ri-transformasie is ondersoek as 'n manier om sekondêre metabolisme te verander om interkloon variasie te skep. Getransformeerde klone kon uitgeken word, aangesien dit meer primêre metaboliete soos sukrose, galaktose en fruktose insluit as die blaar ekstrakte. Hierdie verskil was nie met die huidige GC-MS metodes so duidelik sigbaar op die sekondêre metabolitiese vlak nie. Oor die algemeen toon ekstraksie met asetoon en methanol dichlorometaan (MetOH: DCM) (1:1 v/v) goeie tot gemiddelde antibakteriese aktiwiteit met die minimum remmende konsentrasie (MIC) waardes van 0.39 tot 0.78 mg ml-1. Die in vitro plant kulture het egter sterker weerstand gebied teen twee Gram-negatiewe bakteriese tipes: Escherichia coli (ATCC 11775) en Klebsiella pneumoniae (ATCC 13883), en teen twee Gram-positiewe bakteriese tipes: Bacillus subtilis (ATCC 6051) en Staphylococcus aureus (ATCC 12600). Die harige wortel ekstrakte het geen aktiwiteit teen die swamme, Fusarium subglutinans (MRC 0115) en Fusarium proliferatum (MRC 6908) getoon nie. Mikro-voortplanting is dus 'n interessante manier om S. runcinata kommersieel te produseer aangeien die plante verbeterde farmalogiese aktiwiteit toon. Die biotegnologiese benadering wat hier toegepas word, is 'n praktiese strategie vir die produksie van geneesmiddels van S. runcinata.

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