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

[摘要] 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.

[发布机构] Stellenbosch University