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Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane
Thesis (MScAgric)--Stellenbosch University, 2019.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2019
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| _version_ | 1867613939879116800 |
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| access_status_str | Open Access |
| author | Le Roux, Wildene |
| author2 | Van der Vyver, Christell |
| author_browse | Le Roux, Wildene Van der Vyver, Christell |
| author_facet | Van der Vyver, Christell Le Roux, Wildene |
| author_sort | Le Roux, Wildene |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2019. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/106913 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:06.995Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/106913 Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane Le Roux, Wildene Van der Vyver, Christell Stellenbosch University. Faculty of AgriSciences. Department of Genetics & Institute of Plant Biotechnology. Sugarcane -- Mutation breeding -- South Africa Sugarcane -- Effect of gamma rays on Sugarcane -- Drought tolerance Chemical mutagenesis -- Environmental aspects Sugarcane -- Planting -- Economic aspects -- South Africa Callus UCTD Thesis (MScAgric)--Stellenbosch University, 2019. ENGLISH ABSTRACT: Sugarcane is an economically important crop plant in South Africa as it serves as the primary source of sugar, fulfilling a major role in job creation, earn revenue via export, and as a value-added lignocellulosic biomass. With the current changes in climate, this crop’ sustainability is threatened by limited water resources. Traditional plant breeding methods in sugarcane are hampered due to its high ploidy count and limitations linked to inflorescence and pollen production. Thus, additional methods have to be used to create new cultivars bearing drought-resistant traits. Mutation breeding through chemical and physical mutagenesis approaches were explored in this study to induce useful genomic changes linked to enhance drought tolerant traits. Gamma radiation was used as a method of physical mutagenesis in which Saccharum spp. hybrid cultivars NCo310 and N58 were irradiated, with dosages between 10-40 Gy, and selected for osmotic tolerance under in vitro conditions. Callus cells from these two cultivars were irradiated, selected and regenerated in vitro on growth media containing the osmoticum, PEG6000, creating a simulated drought environment in the hope to obtain plantlets which display an enhance drought tolerance phenotype. One plantlet survived the selection regime but proved unable to survive under normal growth parameters in the glasshouse. Radiation-induced damage was assessed by detecting and monitoring the formation of micronuclei in the irradiated sugarcane cells. Micronuclei have been considered a genotoxic marker for radiation damage in other species, but have never been shown in sugarcane. In most cases, micronuclei formation is known to be directly proportional to the amount of radiation exposure. In this study, microcnuclei were successfully detected through fluorescent microscopy using DAPI as a stain. However, in this study a linear correlation between the radiation dose and amounts of micronuclei formed could not be established. The formation of these micronuclei varied across radiation dosage and detection time after exposure, in comparison no micronuclei were detected in non-irrradiated control sugarcane tissue. EMS (ethyl methanesulphonate) was used as a chemical mutagen in a sugarcane mutation breeding study conducted by Masoabi et al. (2017), which generated a number of in vitro osmotic tolerant plants. In the current study, three of these plantlets were multiplied in vitro and glasshouse pot trials setup. The aim of the experiment was to determine whether the in vitro osmotic tolerant plants also display an enhanced ex vitro drought tolerant phenotype and normal growth attributes. Plants of the M3 mutant line were significantly taller and had more sucrose and glucose in mature stalk tissue when compared to the wildtype control plants. Under drought conditions, all mutant lines survived longer than the N19 WT (wildtype) plants. Physiological and biochemical analysis of the droughted mutant plants further detected higher relative water content in leaves, stomatal conductance and chlorophyll fluorescence suggested that the photosynthetic machinery stayed active longer, less reactive oxygen species formed and higher levels of catalase and superoxide dismutase were present in the mutant lines, when compared to the WT plants. Also, malondialdehyde levels was lower in mutant lines, which contribute to less lipid peroxide damage. These experiments showed promising results in terms of using a chemical such as EMS to induce drought tolerance in sugarcane. AFRIKAANSE OPSOMMING: Suikerriet is ‘n ekonomies belangrike gewasplant in Suid-Afrika aangesien dit dien as die primêre bron van suiker, dit speel ‘n belangrike rol in werkskepping, inkomste verdien via uitvoere, en as ‘n toegevoedge waarde in lignosellulose biomassa. Met die huidige verandering in klimaat word hierdie gewas se volhoubaarheid bedreig deur beperkte waterhulpbronne. Tradisionele planteteling metodes in suikerriet word bemoeilik as gevolg van hierdie gewas se hoë ploїdietelling en beperkings in terme van bloeiwyse en stuifmeel produksie. Dus moet bykomende metodes gebruik word om nuwe kultivars te kweek wat droogte verdraagsame eienskappe toon. Mutasieteling deur middel van chemiese en fisiese mutagenese word in hierdie studie ondersoek om nuttige genomiese veranderinge aan te bring wat gepaard gaan met droogteverdraagsame eienskappe. Gammastraling is gebruik as ‘n metode van fisiese mutagenese waarin die Saccharum spesie hibried kultivars NCo310 en N58 bestraal is, met dosisse tussen 10-40 Gy, waarna seleksie toegepas is onder in vitro toestande vir osmotiese toleransie. Kallus selle van die twee kultivars is bestraal, geselekteer en gegenereer in vitro op groeimedia wat die osmotikum PEG6000 bevat. Die osmotikum simuleer ‘n droogtestres omgewing en die eksperiment is uitgevoer in die hoop om plantjies te verkry wat ‘n droogteverdraagsame fenotipe toon. Slegs een plantjie het die seleksie proses oorleef maar kon nie onder normale groeiparameters in die glashuis oorleef nie. Stralingsgeïnduseerde skade is beoordeel deur die opsporing en monitering van die vorming van mikrokerne in bestraalde suikerriet selle. Mikrokerne word beskou as ‘n genotoksiese merker vir bestralingsskade in ander spesies maar is nog nooit in suikerriet getoon nie. In die meeste gevalle is mikrokernvorming bekend dat dit direk eweredig is aan die hoeveelheid bestraling waaraan die monsters blootgestel is. In hierdie studie is mikrokerne suksesvol geidentifiseer deur fluoreseerende mikroskopie deur die gebruik van DAPI as ‘n kleurmiddel. Daar kon egter nie ‘n lineêre korrelasie tussen die bestralingsdosis en die hoeveelheid mikrokerne wat vorm getrek word nie. Die vorming van mikrokerne het verskil na gelang van die bestralingsdosis en opsporingstyd na blootstelling. In vergelyking is geen mikrokerne bespeur in nie-bestraalde kontrole suikerrietmonsters. EMS (etiel-metaansulfonaat) was as ‘n chemiese mutageen in ‘n suikerriet-mutasieteling studie wat deur Masoabi et al. (2017) uitgevoer is, gebruik. Hierdie studie het ‘n aantal in vitro plantjies wat osmotiese verdraagsaamheid toon gegenereer. In die huidige studie is drie van hierdie plante in vitro vermenigvuldig en gebruik om glashuispotproewe op te stel. Die doel van die eksperiment was om te bepaal of die in vitro-osmotiese tolerante plante ook ‘n verbeterde ex vitro droogtetolerante fenotiepe en normale groei-eienskappe toon. Plante van die M3-mutantelyn was aansienlik groter en het meer sukrose en glukose gehad in volwasse stamweefsel in vergelyking met die wildetipe kontrole plante. Onder droogtestres toestande het alle mutante lyne langer oorleef as die N19 wildetipe plante. Fisiologiese en biochemiese analise van die droogte-gestresde mutante plante het verder getoon dat hoër relatiewe waterinhoud in blare, stomatale geleiding en chlorofil fluoresensie moontlik aandui dat die fotosintetiese masjinerie langer aktief gebly het as in die kontrole plante. Minder reaktiewe suurstofspesies gevorm en hoër vlakke katalase en superoksied dismutase was teenwoordig in die mutante lyne, in vergelyking met die WT plante. Laastens, malondialdehiedvlakke (MDA) was ook laer in gemuteerde lyne wat bydra tot minder lipiedperoksied skade. Hierdie eksperimente het belowende resultate getoon in terme van die gebruik van ‘n chemikalieë soos EMS om droogtetoleransie in suikerriet te induseer. Masters 2019-10-29T13:32:35Z 2019-12-11T06:38:44Z 2021-02-01T03:00:12Z 2019-12 Thesis http://hdl.handle.net/10019.1/106913 en_ZA Stellenbosch University application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Sugarcane -- Mutation breeding -- South Africa Sugarcane -- Effect of gamma rays on Sugarcane -- Drought tolerance Chemical mutagenesis -- Environmental aspects Sugarcane -- Planting -- Economic aspects -- South Africa Callus UCTD Le Roux, Wildene Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| title | Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| title_full | Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| title_fullStr | Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| title_full_unstemmed | Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| title_short | Induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| title_sort | induced mutagenesis through gamma irradiation of embryogenic callus and selection for drought tolerance in sugarcane |
| topic | Sugarcane -- Mutation breeding -- South Africa Sugarcane -- Effect of gamma rays on Sugarcane -- Drought tolerance Chemical mutagenesis -- Environmental aspects Sugarcane -- Planting -- Economic aspects -- South Africa Callus UCTD |
| url | http://hdl.handle.net/10019.1/106913 |
| work_keys_str_mv | AT lerouxwildene inducedmutagenesisthroughgammairradiationofembryogeniccallusandselectionfordroughttoleranceinsugarcane |