Browsing by Author "Magangana, Tandokazi"

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    In vitro growth and development of the sweet medicinal plant Stevia rebaudiana Bertoni
    (Stellenbosch : Stellenbosch University, 2017-03) Magangana, Tandokazi; Makunga, Nokwanda P.; Stellenbosch University. Faculty of Science. Dept. of Botany and Zoology.
    ENGLISH ABSTRACT: Stevia rebaudiana Bertoni is a plant native to the Amambay region located north-east of Paraguay in South America. S. rebaudiana is a natural, sweet perennial herb that contains ent-kaurene diterpene glycosides in its leaves. There are over 9 ent-kaurene diterpene glycosides and stevioside is the most abundant but rebaudioside A is the sweetest. S. rebaudiana also commonly known as Stevia is recognized to have great economic and scientific value around the world due to its sweetness and reported therapeutic properties. As a result, it is cultivated commercially in certain parts of the world. This, however, excludes southern Africa. South Africa has an opportunity to cultivate S. rebaudiana as a new crop for the agricultural sector. The aim of this study was to establish a protocol to determine the best treatment for optimal seed germination using acid scarification, smoke-water, a combination of acid scarification and smoke-water and gibberellic acid. To study the macronutritional requirements of S. rebaudiana plants utilizing nitrogen and phosphate manipulation in vitro. To determine if in vitro derived plant extracts differ in metabolite profiles regarding the main bio-actives (diterpene glycosides) using a metabolomic approach that involved the application of LC-MS and GC-MS technology. To determine the effects of drought and salinity stress on the growth of S. rebaudiana using different concentrations of (w/v) polyethylene glycol 6000 (PEG 6000) and sodium chloride (NaCl) as osmotica. This plant exhibits a low seed germination rate which is a great challenge towards large scale propagation thus making its production expensive. Using a tissue culture system as a propagation study tool, germination of Stevia seeds was tested using 1% (w/v) 2, 3, 5-triphenyl tetrazolium chloride solution in this study. This showed a low viability of 19%. S. rebaudiana seeds were subjected to four variables namely: smoke water extract, chemical scarification using 70% (v/v) sulfuric acid for 30 seconds, a combination of smoke water extract and 70% (v/v) sulfuric acid and gibberellic acid were tested as a means of improving germination in vitro. The smoke treatment was highly efficacious in producing a significant germination percentage (P < 0.05) while seeds scarified using 70% (v/v) H2SO4 had the lowest germination rate. To test the effect of macronutrients (nitrogen and phosphate), various levels of nitrogen and phosphate were added to the growth medium. Thereafter, liquid chromatography-mass spectrometry was used to analyze the effects on the metabolomic profile. All other 85 nutritional elements were kept similar to the control which contained similar concentrations as Murashige and Skoog (1962) medium (MS) with both nitrogen (NH4NO3 at 20.61 mM and KNO3 at 18.79 mM) and phosphate (KH2PO4 at 1.25 mM). Two distinct clusters were revealed after principal component analysis of the metabolite profiles. The orthogonal partial least squares discriminant analysis was also applied. This allowed the organization of the clusters into two distinct groups. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the distinct separation of phosphate-treated plants from the nitrogen-treated plants. The clustering suggests different chemical influences at enzyme and gene level on secondary metabolism resulting in different chemical profiles. Reducing the nitrogen level to half (0.5 N) in the MS medium led to the tallest plants. Reduction in the roots was observed with increasing levels of nitrogen and phosphate. I further assessed the effects of drought and salinity stress by using polyethylene glycol 6000 (PEG) and sodium chloride (NaCl) at different concentrations, respectively. Higher concentrations of PEG 6000 (7.5 and 10%) and NaCl (75 and 100 mM) resulted in a decline in both ent-kaurene diterpene glycosides and terpenes present in the treated Stevia leaves. Headspace solid phase microextraction gas chromatography spectrometry revealed an abundance of α-pinene, β-pinene and sabinene in all treated plants except in the plants exposed to 10% PEG 6000 which showed no growth. The addition of PEG 6000 decreased the concentrations of rebaudioside A and stevioside significantly. In conclusion, this study has revealed the importance of nitrogen and phosphate in the manipulation of ent-kaurene diterpene glycoside production in Stevia microplants, setting a platform to test these effects ex vitro.