Towards understanding the metabolism of in vitro Sutherlandia frutescens (L.)R.Br. cultures

[摘要] 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 havetraditionally been used for the treatment of various diseases. Reports have also indicated thatS. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, severalflavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to themedicinal properties of this plant.The first part of this study involved testing the effect of six treatments (light, dark, soaking ofseeds, 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 seedsgerminating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledonsand 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 furtherstudies. It would be possible to successfully adopt it for commercial production of plants toassist with cultivation of Sutherlandia as a field crop. Another advantage of this system isspontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. Theserooted plants were acclimated in the glasshouse using vented lids to harden off the shoots andthis 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 threeAgrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient atinducing hairy roots in both hypocotyls and cotyledons. However, transformation of S.frutescens was dependent on the type of explant used with the hypocotyls being moreefficiently 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 cloneswere selected and their cultivation in a liquid system was optimized by investigating theirgrowth 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 thegrowth of hairy root clones was best in the B5 and MS medium, with White's medium being theleast effective cultivation medium. Molecular analysis of hairy roots was used to prove thetransgenic status of these four putative transgenic clones. This was achieved usingpolymerase 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 Southernhybridization a radioactively labeled rol A probe was used to determine the copy number of therol 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 (1xnitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice thenormal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparaginelevels in the roots and shoots and the canavanine level in the shoots were directly correlatedwith the amount of nitrogen in the medium (as the nitrogen level increased, the metabolitelevels increased). The GABA level in the shoots was inversely correlated with the amount ofnitrogen in the medium. Several reasons may explain these metabolic changes including theassimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. Thereduced GABA levels may indicate the preferential flux of the free GABA into other nitrogenassimilatory pathways such as protein synthesis as well as its rapid utilization to replenish thetricarboxilic 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 theshoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did notsignificantly 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. Saltstress only resulted in a significant increase in arginine levels with increasing salinity and alsocaused a reduction in the rooting and growth response. Lowered plant vigour may be the firstvisual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmoticadjustment resulting in changes at the metabolic level concomitant to physiological growthchanges.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 andStaphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots wereinvestigated using TLC and LC-MS analysis. Several of the compounds in the hairy rootextracts were present in higher levels than in the control root extracts. Transformation alsoincreased the complexity of the phytochemical pattern of the hairy roots, either due thesynthesis of novel compounds or upregulated synthesis of existing metabolic pathways. Theproduction of hairy roots and the establishment in a liquid system during this study was animportant step towards upscaling these cultures to a bioreactor. In future these roots canassist in developing cultures which produce a high yield of the desired metabolites.

[发布机构] Stellenbosch University