Browsing by Author "Makunga, Nokwanda P."
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Item Genetic modification in Pinus patula using transgenic technology.(2006) Nigro, Sara Anna.; Van Staden, Johannes.; Makunga, Nokwanda P.; Jones, Nicky B.Progress in tree biotechnology initially trailed behind agricultural crops due to their long life cycle, difficult tissue culture and regeneration protocols, and their abundance in natural forests. However, rapid global deforestation rates, together with an increased world demand for pulp, paper and timber products, have prompted scientific and commercial focus to improve genetic timber stocks. South Africa, a tree-poor country (where indigenous forests are protected), has relied almost solely on exotic plantations to meet its demand for timber. A pioneer study investigating the feasibility of using direct (biolistic) and indirect (Agrobacterium-mediated) methods for gene transfer was undertaken in Pinus patula Schiede et Deppe, a Mexican softwood and a forerunner for saw timber, pulpwood and paper in the South African forest industries. The aim of the transformation methods was to impart herbicide resistance to the trees. This was achieved via the introduction of a bar-GUS pAHC25 cassette under the control of the ubiquitin promoter. To provide target material for transformation, two in vitro micropropagation pathways were used: somatic embryogenesis and organogenesis. Both embryonal suspensor masses (ESM) and somatic embryos at various stages of development were initially used as target explants for the biolistic study using an established in vitro protocol. A stepwise selection was implemented in order to allow transformed (particularly bombarded) cultures the opportunity to regenerate under selection pressure using MSG3 maintenance medium supplemented with BASTA® herbicide at 1 mg l ¯¹ followed by 3 mg l¯¹ active ingredient at the next subculture. Biolistic transgene delivery was more efficient when sorbitol was included in the pre-bombardment medium enabling use of higher vacuum and shooting pressures, without lowering the regeneration potential of ESM significantly. Bombarded material from two genotypes (Lines 2 and 3) was regenerated to produce mature somatic embryos using an optimized regeneration regimen. The indirect study with Agrobacterium tumefaciens (LBA4404), transformed with the pAHC25 vector via triparental mating or heat shock, used a variety of target tissues including: mature somatic embryos, ESM and mature zygotic embryos (MZE's) - a novel in vitro system for P. patula. The Agrobacterium-mediated method resulted in optimized decontamination conditions using a combination of liquid MSG3 (or sterile dH₂O for mature embryos) supplemented with 500 mg l ¯¹ cefotaxime, with rotation, and sterile 65 mm Whatman No. 3 filter paper stacks, which avoided excess filtering and stress to transformation material. Further efforts to aid regeneration during the indirect study included L-proline post-transformation, though no mature somatic embryos were regenerated at the conclusion of the Agrobacterium-mediated study. Recovery of transformed ESM in both studies was best during the active growth phase 4-6 d after subculture. Regeneration with good somatic embryo potential was an exigent aspect in both transformation studies. Expression of positive histochemical GUS activity in all transformed material was confirmed by polymerase chain reaction (PCR) analysis indicating that Pinus patula tissue was amenable to transformation. A new bar PCR regime was implemented in P. patula. In the biolistic study, a higher transformation efficiency of bar amplicons (53%) than GUS amplicons (45%) was observed, reflecting their non-linked status on the pAHC25 transformation vector. This is the first report of biolistic transformation of P. patula that will allow for the production of transgenic ESM. The production of transgenic P. patula holds great promise for commercial development in the South African forestry industry. The application of transgenic trees in the timber industry is numerous but the aims most relevant to P. patula include wood modification and disease resistance to pathogens like pitch canker fungus.Item Genetic transformation and micropropagation of Thapsia garganica L. - a medicinal plant.(2003) Makunga, Nokwanda P.; Van Staden, Johannes.; Jäger, Anna Katharina.No abstract available.Item In vitro conservation of endangered Dierama species.(2004) Madubanya, Lebogang Angelo.; Fennell, Catherine W.; Makunga, Nokwanda P.; Van Staden, Johannes.No abstract available.Item Molecular basis of anthocyanin production in callus and cell cultures of Oxalis reclinata.(1996) Makunga, Nokwanda P.; Van Staden, Johannes.; Cress, William A.Oxalis reclinata Jacq., is a dicotyledonous plant. O. reclinata belongs to the family Oxalidaceae. This plant produced callus which accumulated red coloured anthocyanin pigments when cultured in vitro. The levels of anthocyanin accumulated by O. reclinata callus were higher than in the intact plant. The major pigment was isolated and identified as cyanjdin-3-glucoside (CROUCH, VAN STADEN, VAN STADEN, DREWES & MEYER, 1993). In nature, anthocyanins are responsible for orange, red, purple and blue colouration of certain tissues of higher plant s. Due to the toxicity of many synthetic red colouring agents, anthocyanins are regarded as potential substitutes for synthetic food colourants. This research was aimed at investigating mechanisms which induce pigment production as well as to optimize anthocyanin yield from callus cultures of O. reclinata, once anthocyanin production was stimulated. Pigmented and non-pigmented callus lines were generated from O. reclinata (CROUCH & VAN STADEN, 1994) and maintained on MURASHIGE & SKOOG (1962) agar medium (O.8% [w/v], pH 5.7) supplemented with 0.5 mgℓ ¯¹ BA, 5 mgℓ ¯¹ NAA, 30 gℓ ¯¹ sucrose and 0.1 gℓ ¯¹ myo-inositol. Plant tissue culture studies were conducted on red and white lines of O. reclinata to optimize callus yield and anthocyanin production in vitro. This involved manipulating contributory factors of the culture environment (carbohydrates, nitrates, phosphates, phytohormones, light and temperature). In vitro studies showed that, light played an inductive role in anthocyanin production in callus cultures of O. reclinata. The auxin, 2,4- dichlorophenoxyacetic acid (2,4-D) reduced pigment production but increased callus biomass. This hormone probably exerted its effect by reducing the pool of anthocyanin precursors, such as phenylalanine, resulting in increased primary metabolic activity. Suspension cultures were shown to be a viable means of propagating pigmented callus cells of O. reclinata. The growth curves for red and white callus cells were determined using the settled cell volume (SCV) method. Pigmented cell cultures grew for longer periods compared to nonpigmented cells of O. reclinata. White callus cells reached the stationary phase after 18 days. Red callus cells continued growing exponentially for an extra three days compared to white callus cells. The vacuole was identified as the organelle where anthocyanins accumulate using the light microscope. The molecular techniques of two-dimensional electrophoresis and in vitro translation were utilized to analyze differences in gene expression between white and red callus cultures of O. reclinata. Thus far, two-dimensional electrophoresis has shown that the red callus of O. reclinata had more polypeptides compared to the white callus. The level of gene expression was higher in the red callus compared to white callus, as revealed by nonradioactive in vitro translation. With optimization of radioactive in vitro translation, identification of specific structural anthocyanin genes which are under regulatory control should be possible. Future research should aim at acquiring a better understanding about the genetic control of anthocyanin biosynthesis in order to manipulate this pathway effectively.