(Triticum aestivum L.)
Transcription
(Triticum aestivum L.)
Diss. ETH No. 12411 Single-Stranded DNA as a Tool for Genetic Transformation in Wheat (Triticum aestivum L.) A dissertation submitted to the SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH for the degree of Doctor of Natural Sciences presented by Murielle UZE DEA de Biologie Moleculaire Strasbourg, born May et Cellulaire France 27 1967 citizen of France accepted on the recommendation of Prof. Dr. I. Potrykus, examiner Prof. Dr. B. Keller, co-examiner PD. Dr. C. Sautter, co-examiner October 1997 II SUMMARY Wheat is much effort has been put into resistance important cereals of of the most one embryonic calli bombardment to can work frequency develop to was well as the as Agrobacterium would method a embryos from immature efficiency which be the system of choice, widely is used to is still low. The aim of transformation the increases of gene transfer for quality disease goal. Currently, contribute to this transform wheat. However, the transformation my (e.g. for wheat genetically improving quality). Gene transfer or the world. Therefore, single copy insertions. unfortunately, its natural host range is restricted to dicot's. However, different labs demonstrated efficient rice by Agrobacterium, indicating transformation Agrobacterium tumefaciens types have been screened on and A. immature that it might rhizogenes work in wheat also. strains of different zygotic embryos opine from several wheat varieties. A T-DNA transfer to wheat cells has been shown in transient with pus-intron regeneration was unsuccessful. In contrast, rice before inoculation to the transfer, play an cell free a Agrobacterium by using {gus and bar) important of bacteria case tried we high efficiency with by Agrobacterium In were cointroduced single- (ss) DNA can ssDNA. or double-stranded integrate be ds or ss might limiting, be we a compared: It was efficiency element partially in rice. for gene the T-DNA of DNA. Two marker genes circular (c) or in wheat linear (I) as discovered that linearized high efficiency of bar and plant plasmolysis by microprojectile bombardment were but have been transformed limiting single-stranded form with l-ssDNA showed the and mimicked (ds) plasmid. Although coexpression of higher for dsDNA, ssDNA as plants role for gene transfer approach linearized the gus gene and I have shown callus representing cells. Four different DNA structures a expressed Some sectors marker gene. a up to 14% for I- gus after cobombardment was higher cotransformation efficiency. Since I- good substrate for degradation, and its nuclear import might suggest agrobacterial proteins to deliver l-ssDNA similar to T-DNA. fragments with VirD2 and VirE2 Ill RESUME plus importantes Le ble est une des cereales au monde. Pour cela, de nombreux efforts sont apportes dans son pour les resistances de maladie qualite nutrionnelle). contribuer peut a zygotiques immatures mon objectifs. ces transformer le ble est travail consistait le developper a efficacement particules son Plusieurs souches une methode il ete a qui augmenterait etre d'opine types transitoirement indiquant d'Agrobacterium tumefaciens ont ete criblees en sur vers des et A. frequence systeme de la classe a transforme se pourrait la cellule evidence par Putilisation du gene ont ete obtenues et j'ai important montre que la plasmolyse rhizogenes embryons aucune a ete mis Des secteurs plante avec de zygotiques vegetale gftvs-intron. regeneree. Au contraire, des plantes de riz transformers role la methode cette que bleus exprimant le gene gus ont ete obtenus mais un le demontre que le riz immatures de ble. Un transfert de T-DNA joue pour embryons des sur hote naturel de plantes est restreint Agrobacterium, par utilisee ble. au differents technique actuellement La copie unique. Agrobacterium pourrait de Le transfert de gene qualite de transfert de gene pour obtenir des dicotyledones. Cependant, s'etendre genetique (par exemple mais le rendement demeure encore faible. Le but de transformation choisi mais des la bombardement de de transformation ainsi que la insertions ou amelioration n'a pu etre Agrobacterium des cals avant I'inoculation pour I'efficacite de transfert de gene dans les cellules de riz. Dans le gene, cas ou seconde approche une d'Agrobacterium (gus et la bacterie serait le facteur limitant pour le transfert de bar) avec de I'ADN a consiste simple a imiter partiellement le T-DNA brin lineaire. Deux marqueurs de gene ont ete cointroduits par cobombardement dans les cellules de ble. Quatre differentes structures de I'ADN ont ete comparees: I'ADN circulaire (c), ou lineaire (I) en tant que DB ou tant que simple (SB) ou double brin (DB). SB, semble integrer le genome bu ble L'ADN lineaire, avec une en grande IV efficacite par rapport des coexpression au circulaire, jusqu'a 13.92% pour le SB. Bien que la genes bar et gus cobombardement, plus elevee que le l-SB, etait ce limitee, degradation nous et que son importation suggerons de delivrer les VirD2 et VirE2 d'Agrobacterium, I'ADN dernier montrait efficacite de cotransformation. Comme I'ADN l-SB pour la pour pourrait vers a une plus apres haute etre un bon substrat le noyau cellulaire serait fragments d'ADN SB similaire l-DB, avec la structure du T-DNA. les proteines