Genetic enhancement of pearl millet

O'Kennedy, Martha Margaretha (2004-03)

Thesis (PhD)--Stellenbosch University, 2004.

Thesis

ENGLISH ABSTRACT: The aim of this study was toe stablish a reliable protocol for the production 0 f transgenic pearl millet as this will open new avenues for augmenting the gene pool of this crop. This was achieved by identifying a highly regenerabie genotype and optimisation of a tissue culture system, and biolistic protocol f or stable integration of selected transgenes. Both a negative, herbicide resistance selectable marker gene, bar, and a positive selectable marker gene, manA, were individually introduced in order to identify and establish a reliable transformation protocol. The optimised transformation protocol was then used to introduce an antifungal gene in the genome of pearl millet to enhance resistance to the biotrophic fungus Sclerospora graminicola. S. graminicola, an obligate oomycetous fungal phytopathogen, is the causal agent of downy mildew in pearl millet plants and a major constraint in the production of pearl millet. A single component of antifungal resistance was introduced into the genome of pearl millet, as preliminary work towards determining its role in the total plant defence system. The approach chosen was to introduce a hydrolytic enzyme, 13-1,3- glucanase, from Trichoderma atroviride (formerly T. harzianum), a soil-borne filamentous fungus, capable of parasitizing several plant pathogenic fungi. It was anticipated that introducing this glucanase gene from T. atroviride which degrades glucan in the fungal cell walls, would significantly contribute to the improvement of resistance against downy mildew. Constructs were prepared containing the gene (gluc78) encoding a 78 kDa beta-1,3- glucanase. The constructs were prepared containing the gluc78 gene driven either by a strong constitutive promoter (ubiquitin promoter, exon and intron) or a wound inducible promoter, the potato proteinase inhibitor ilK gene promoter. The wound inducible promoter includes either an AMV leader' sequence or the rice Act1 intron to obtain higher expression levels in the monocotyledonous plant. The transformation efficiency using the particle inflow gun and the herbicide resistance gene, bar, was improved from 0.02% on a MS based medium, to 0.19 or 0.72% with manA as selectable marker gene on MS or L3 based medium, respectively. However, individual experiments, introducing manA as selectable marker gene, resulted in frequencies of 1.2 and 3%. This translated to one transformation event per plate, which contains on average 31-35 pre-cultured immature zygotic embryos. This is the first report of t he successful introduction and expression of a 13-1,3-glucanase encoding gene from a biocontrol fungus not only under constitutive expression but also under wound inducible expression in a plant. Optimisation of genetic engineering of pearl millet, a cereal crop recalcitrant to transformation, and the introduction of an antifungal transgene, was accomplished in this study. Initial results hint that expression of this transgene enhances resistance to S. graminicola.

AFRIKAANSE OPSOMMING: Die doel van die studie was om 'n betroubare genetiese transformeringsprotokol vir pêrel manna te ontwikkel. Hiervoor moes eerstens 'n regenereerbare genotipe geidentifiseer word. Twedens moes 'n betroubare weefselkultuur en biolistiese transformeringssisteem ontwikkel word. Beide die onkruiddoder bestandheidsgeen, bar, en 'n positiewe selektiewe geen, manA, is vir die doel van die projek onafhanklik in die genoom van pêrel manna in gekloneer. Die optimale sisteem is vervolgens aangewend om 'n geen wat potensieël verbeterde bestandheid teen die biotrofiese swam Sclerospora graminicola wat donsige meeldou by plante veroorsaak, in pêrel manna in te kloneer. 'n Enkele komponent van bestandheid is in die genetiese material van pêrel manna in gekloneer as inleidende werk om die rol van hierdie geen in die totale verdedigingsisteem te bepaal. Die benadering wat gekies was, behels die klonering van 'n hidrolitiese ensiem 13-1,3-glukanase, van Trichoderma atroviride (voorheen T. harzianum), 'n grondgedraagde swam, wat op 'n aantal ander plantpatogene fungus kan parasiteer. Die verwagting is dat klonering van hierdie 13- 1,3-glukanase geen van T. atroviride wat die glukaan verteer in die selwande van swamme, 'n groot verbetering tot die bestandheid teen donsige meeldou sal meebring. Konstrukte is voorberei wat die gluc78 geen bevat wat kodeer vir die 78 kDa beta-1,3-glukanase protein. Die konstrukte wat voorberei is bevat die gluc78 geen geinduseer deur of 'n sterk konstituwe promoter (ubiquitin promoter, exon en intron) of deur 'n wond geinduseerde promoter, die aartappel proteinase inhibeerder ilK geen promoter. Hierdie promoter word gevolg deur of 'n AMV leier volgorde of die rys Act1 intron om verhoogde uitdruk vlakke in monokotiele plante te verseker. As die partikel invloei geweer in kombinasie met die onkruiddoderbestandheidsgeen gebruik word, was die doeltreffendheid van transformasie 0.02% op 'n MS gebasseerde groeimedium. 'n Transformasie doeltreffendheid van onderskeidelik 0.19 en 0.72% is verkry wanneer die manA as selektiewe geen gebruik is op MS of L3 gebasseerde medium. Twee individual eksperimente, waar die manA geen as selektiewe geen gebruik is, het gelei tot 'n transformasie doeltreffendheid van 1.2 of 3%. Dit gee 'n gemiddelde van een transformasie per plaat wat 31 tot 35 voorafgekweekte onvolwasse embrios bevat. Hierdie is d ie eerste verslag van d ie suksesvolle klonering en uitdrukking van 'n 13-1,3-glukanasekoderende geen van 'n swam wat as 'n biologiese beheeragent gebruik word. Hierdie is nie alleenlik onder konstitutiewe uitdrukking nie, maar ook 0 nder wond g einduseerde u itdruk in' n p lant. In hierdie studie is die 0 ptimisering van genetiese verbetering van pêrel manna, 'n graan gewas wat gehard is teen transformasie, deur die klonering van 'n bestandheidsgeen in die genoom van hierdie gewas gedoen. Aanvanklike resultate dui daarop dat die uitdruk van hierdie geen lei tot verbeterde bestandheid teen S. graminicola.

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