Free Access
Volume 12, Number 7, 1992
Page(s) 537 - 544
Agronomie 12 (1992) 537-544
DOI: 10.1051/agro:19920705

Investigations on transforming Triticum aestivum via the pollen tube pathway

N. Martin, P. Forgeois and E. Picard

INRA-UPS-CNRS, Station de Génétique Végétale, Ferme du Moulon, 91190, Gif-sur-Yvette, France

Abstract - Attempts were made to optimize the necessary conditions to transform wheat Triticum aestivum using the pollen tube pathway. Three methods previously described as successful in cereals and plasmid constructions carrying either NPT II or GUS markers were tested. Functionality of GUS expression vector was checked by particle gun experiments on wheat leaves. Experiments described in this paper show that a Tris-HCl - 150 mM pH 9 solution containing 0.2 M MgSO4 inhibits both stigma and pollen nucleases. However, none of the tested plants among the 2 731 screened for GUS activity or for their resistance to kanamycin, expressed the reporter genes.

Résumé - Recherches sur la transformation du blé Triticum aestivum par la voie du tube pollinique. Nous avons tenté d'optimiser les conditions nécessaires pour transformer le blé Triticum aestivum en utilisant la voie du tube pollinique. Trois méthodes ayant été décrites comme des succès chez les céréales et des constructions possédant soit les gènes marqueurs NPTII ou GUS ont été testées. La fonctionnalité de l'expression du gène GUS a été contrôlée au moyen d'un canon à particules sur des feuilles de blé. Les expérimentations décrites montrent que la solution Tris-HCl 150 mmol·l-1 pH9 contenant 0,2 mol·l-1 de MgSO 4 inhibe les nucléases du pollen et des stigmates. Toutefois aucune des 2 731 plantes testées pour leur activité GUS ou leur résistance à la kanamycine n'a exprimé ces gènes marqueurs.

Abstract - INTRODUCTION Among the cereals, wheat and barley remain the most difficult to transform, mainly because the regeneration step is limiting when in vitro methods are used in transformation experiments. The pollen tube pathway has been considered to be a promising method for transformation of the zygote in vivo. Three methods have been mentioned in the literature. The use of pollen as a DNA vector was first proposed by De Wet et al (1985). In the methods used, pollen grains were incubated in a solution containing exogenous genetic material and then used for pollination. Ohta (1986) and Hess (1987) reported a highly efficient transformation system using a similar method in maize and Nicotiana glauca, but without molecular evidence. In these cases 2 explanations for the exogenous DNA uptake by pollen have been suggested: i), the intine of a dry pollen could be leaky for a short time immediately after deposition on the stigmas. During this leaky phase, macromolecules could pass through the intine (Heslop-Harrison, 1979); ii), DNA could be taken up by the tip of the growing pollen tube which lacks cell walls (De Wet et al, 1985). Abdul-Baki et al (1990) demonstrated that the pollen of Nicotiana gossei can incorporate labelled DNA in a pollen germination medium (PGM). Futhermore, electroporation can enhance the percentage of DNA uptake. Aokas (1987) used liposomes to facilitate DNA uptake and demonstrated the incorporation of labelled DNA by pollen. These results confirmed that the pollen tube can be considered as a protoplast near

Key words: Triticum aestivum = wheat / transformation / pollen / nuclease

Mots clés : Triticum aestivum = blé / transformation / pollen / nucléase