In vitro growth and development of the sweet medicinal plant Stevia rebaudiana Bertoni

Thesis (MSc)--Stellenbosch University, 2017.

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.

AFRIKAANSE OPSOMMING: Stevia rebaudiana Bertoni is ‘n inheemse plant van die Amambay streek, Noord-Oos van Paraguay in Suid-Amerika. S. rebaudiana is ‘n natuurlike soet, meerjarige krui wat ent-kaurene diterpeen glikosiedes bevat in die blare. Daar is meer as 9 ent-kaurene diterpeen glikosiedes waarvan steviosied die mees volop is, maar rebaudiosied A is die soetste. S. rebaudiana (Stevia) is bekend vir sy merkwaardige ekonomiese en wetenskaplike waarde wêreldwyd, as gevolg van sy soetheid en berigte terapeutiese eienskappe. As gevolg word dit kommersiëel vervaardig in sekere dele van die wêreld. Hierdie sluit alhoewel nie Suid-Afrika in nie, en dus is daar ‘n geleentheid vir vervaardiging van S. rebaudiana as ‘n nuwe kropgewas in die lanbousektor. Die doel van hierdie studie was om 'n protokol op te stel vir die beste behandeling vir optimale ontkieming met behulp van suurbeskadiging, rookwater, 'n kombinasie van suurbeskadiging en rookwater en gibberelliensuur. Om die makronutriënt vereistes van S. rebaudiana plante te bestudeer deur gebruik te maak van in vitro stikstof en fosfaat manipulasie. Om te bepaal of plant ekstrakte wat in vitro versamel is, verskil in metaboliet profiele met betrekking tot die belangrikste bio-aktiewe molekules (diterpeen glikosiede) met behulp van 'n metabolomiese benadering wat die toepassing van LC-MS en GC-MS tegnologieë gebruik. Om die uitwerking wat droogte en soutgehalte stres het op die groei van S. rebaudiana te bepaal met behulp van verskillende konsentrasies van (w/v) poliëtileenglikol 6000 (PEG 6000) en natriumchloried (NaCl) as osmotika. Hierdie plant het laë ontkiemings-sukses, wat ‘n groot uitdaging is vir groot-skaal boerdery, en as gevolg produksie duur maak. Na aanvang van ‘n tetrasolium toets met gebruik van 1% (w/v) 2, 3, 5-trifeniel tetrasolium chloried oplossing, was ‘n weefselkultuur sisteem gebruik as ‘n wetenskaplike werktuig om ontkieming-sukses van Stevia saad te toets. Lewensvatbaarheid na hierdie toets was slegs 19%. S. rebaudiana sade was behandel met vier veranderlikes naamlik: rookwaterekstrak; chemiese skade met gebruik van 70% (v/v) swaelsuur vir 30 sekondes; ‘n kombinasie van rookwaterekstrak en 70% (v/v) swaelsuur and gibberelliensuur. Hierdie veranderlikes was getoets as ‘n metode vir die verbetering van in vitro ontkieming. Die rook behandeling was hoogs effektief met ‘n beduidende ontkieming persentasie (P < 0.05), terwyl saaddoppe wat verswak was met 70% (v/v) swaelsuur, die laagste ontkiemings-sukses gehad het. Om die effek van makronutriënte te toets, was verskeie vlakke van stikstof en fosfaat by die groeimedium gevoeg. Dit was gevolg deur vloeistofchromatografie-massaspektrometrie te gebruik om die effekte op die metaboliese profile te ontleed. Al die ander 85 voedingstowwe was behou soortgelyk aan die kontrole, wat dieselfde konsentrasie Murashige and Skoog (1962) medium (MS) met albei stikstof (NH4NO3 van 20.61 mM en KNO3 van 18.79 mM) en fosfaat (KH2PO4 van 1.25 mM). Twee onderskeie groepe was merkbaar na hoofkomponentanalise van die metaboliese profiele. Die ortogonale gedeeltelike kleinste kwadrate diskriminantontleding was ook gebruik. Dit het die groepering van twee onderskeie groepe moontlik gemaak. Steviol hidraat, steviosied hidraat en rebaudiosied A het beduidende bedrae tot die verdeling van fosfaat-behandelde plante van die stikstof-behandelde plante. Die groepering stel verskeie chemiese invloede op ensiem en geen vlak vir sekondêre metabolism, wat lei tot unieke chemiese profiele. Die halvering van die stikstof konsentrasie (0.5 N) in die MS medium, het gelei tot die hoogste lengte plante. Verminderde wortellengte was waargeneem deur verhoogde vlakke van stikstof en fosfaat. Die effekte van droogte en soutgehalte stres was geëvalueer deur gebruik van poliëtileenglikol 6000 (PEG) en natriumchloried (NaCl) teen verskeie konsentrasies, onderskeidelik. Hoër konsentrasies van PEG 6000 (7.5 en 10%) en NaCl (75 en 100 mM) het gelei tot ‘n vermindering in beide ent-kaurene diterpeen glikosiedes en terpene teenwoording in Stevia blare. Kopspasie vastestoffase mikroekstraksie gaschromatografie spektrometrie het ’n oorvloed van α-pineen, β-pineen en sabineen in alle behandelde plante waargeneem behalwe dié blootgestel aan 10% PEG 6000, wat geen groei aangetoon het nie. Die byvoeging van PEG 6000 het die konsentrasies van rebaudiosied A en steviosied drasties verminder. Hierdie studie het die belang van stikstof en fosfaat in die manipulasie van ent-kaurene diterpeen glikosied vervaardiging in Stevia mikroplante bewys, wat ‘n platform skep om hierdie effekte ex vitro te toets.