ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have
traditionally been used for the treatment of various diseases. Reports have also indicated that
S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several
flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the
medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of
seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds
germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons
and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However,
this PGR-free system successfully provided a continuous supply of plant material for further
studies. It would be possible to successfully adopt it for commercial production of plants to
assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is
spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These
rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and
this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S.
frutescens using Agrobacterium-mediated transformation. The efficiency of three
Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at
inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S.
frutescens was dependent on the type of explant used with the hypocotyls being more
efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl
(93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones
were selected and their cultivation in a liquid system was optimized by investigating their
growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White,
1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the
growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the
least effective cultivation medium. Molecular analysis of hairy roots was used to prove the
transgenic status of these four putative transgenic clones. This was achieved using
polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp)
genes to determine the presence of the TL-DNA in the plant genome. During Southern
hybridization a radioactively labeled rol A probe was used to determine the copy number of the
rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x
nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the
normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine
levels in the roots and shoots and the canavanine level in the shoots were directly correlated
with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite
levels increased). The GABA level in the shoots was inversely correlated with the amount of
nitrogen in the medium. Several reasons may explain these metabolic changes including the
assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The
reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen
assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the
tricarboxilic acid cycle intermediates.
The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress
(induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the
shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not
significantly alter the metabolite levels, but resulted in a significant decrease in the growth
(fresh weight and total shoot length) and the rooting response of these microshoots. Salt
stress only resulted in a significant increase in arginine levels with increasing salinity and also
caused a reduction in the rooting and growth response. Lowered plant vigour may be the first
visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic
adjustment resulting in changes at the metabolic level concomitant to physiological growth
changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and
Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were
investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root
extracts were present in higher levels than in the control root extracts. Transformation also
increased the complexity of the phytochemical pattern of the hairy roots, either due the
synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The
production of hairy roots and the establishment in a liquid system during this study was an
important step towards upscaling these cultures to a bioreactor. In future these roots can
assist in developing cultures which produce a high yield of the desired metabolites.
AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige
meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is
tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui,
dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n
Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter
triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene
(alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur
(GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae.
Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade,
fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van
Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te
identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die
saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die
fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae.
Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek
was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in
die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem
suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou
egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële
produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n
Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro
mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante
was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel
afharding) en het tot ‘n 100% oorlewing gelei.
Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met
behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie
Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante
(kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om
harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk
egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as
die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel
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(47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone
was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei
in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934;
White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5
en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s
medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels
was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te
bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en
C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon.
Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A
geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone.
Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore
(stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS,
asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet
produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels
asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die
mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was
aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS
medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee
keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en
asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was
positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die
stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie
redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die
assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die
verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende
metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS
ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul.
Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en
sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die
stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel
kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring
nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en
bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle
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toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die
toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele
teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei
en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die
metaboliet vlakke asook veranderinge in fisiologiese groei, lei.
Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die
transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal
ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige
anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella
pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon.
Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag
chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise
ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in
die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels
se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of
weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels
en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n
belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan
toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.
