Engineering wheat (Triticum aestivum L.) for abiotic resilience by manipulating small ubiquitin-like modifiers

Le Roux, Marlon Luke (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Research is becoming increasingly more focused on innovative strategies to improve wheat (Triticum aestivum L.) to meet current and future consumer demands. With the number of countries facing extreme climate variability increasing, drastic steps need to be taken to ensure local food security. Although genetic engineering of staple crops has been explored widely, they require challenging platforms with highly efficient explants with enhanced regenerative abilities for callus formation and somatic embryogenesis to consistently yield plantlets with altered attributes. Thus, this study describes the necessary steps to obtain transgenic wheat with relative ease, with the goal of improved abiotic stress tolerance. The developed in vitro regeneration and cryopreservation system was then used to introduce genes into bread wheat, namely Overly Tolerant to Salt 1 (OTS1) and OTS2 (both are Small Ubiquitin-like Modifier (SUMO) protease genes) and transcription factor known as Inducer of CBF expression 1 (ICE1). The class of molecules have emerged as an influential mechanism for targeted protein management, as SUMO proteases play a vital role in regulating pathway flux and are therefore ideal targets for manipulating stress-responsive SUMOylation. This study thus describes the isolation and cloning of three genes and its manipulation into constitutive and drought inducible vectors. The latter makes use of a unique promoter which was characterized in silico, after which it was then applied. Finally this study also demonstrated the compatibility of the vectors within wheat as it was confirmed that genetic modification of wheat was achieved by particle bombardment.

AFRIKAANSE OPSOMMING: Navorsing lê toenemend meer klem op die ontwikkeling van innoverende strategieë met die oog op die verbetering van koring (Triticum aestivum L.) opbrengste om aan die huidige sowel as die toekomstige behoeftes te voldoen. Met die toenemende klimaat veranderinge wat talle lande in die gesig staar, moet drastiese stappe geneem word om plaaslike voedselsekuriteit te verseker. Alhoewel die genetiese modifisering van stapelvoedsel gewasse al wyd verken is, is daar ‘n behoeftes vir uitdagende platforms met die vermoë om begin material (bv. onvolwasse embrios) doeltreffend om te skakel in kallus en somatiese embrios om ten einde deurlopend plante met verbeterde eienskappe te lewer. Dus, die huidige studie beskryf die noodsaaklike stappe wat gevolg moet word om transgeniese koring met relatiewe gemak te verkry met die doel om strestolerasie te verbeter. Die ontwikkelde in vitro sisteem, was aangewend om bepaalde gene, naamlik “Overly Tolerant to Salt 1” (OTS1) and OTS2 (beide SUMO protease gene), in broodkoring uit te druk. Dieselfde stappe is gevolg met ‘n addisionele transkripsie factor genaamd “Inducer of CBF expression 1” (ICE1). Die SUMO (“Small Ubiquitin-like Modifier”) klas van molekule het na vore gekom as ‘n invloedryke meganisme vir geteikende proteinbestuur, aangesien SUMO protease ‘n noodsaaklike rol speel in die regulering van biochemiese pad verloop en is dus ideale teikens vir die manipulering van stress-verwante SUMO-lasie. Hierdie studie handel dus oor die isolasie en klonering van drie gene en hul manipulasie in konstitutiewe en droogte induserende vektore. Laasgenoemde maak gebruik van ‘n unieke promoter wat in silico gekarakteriseer is. Laastens word die bruikbaarheid van die vektore in koring gedemonstreer nadat suksesvolle genetiese modifisering bevestig is.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98275
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