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Ann. For. Sci.
Volume 59, Number 5-6, July-October 2002
Proceedings of the Wood, Breeding, Biotechnology and Industrial Expectations Conference
Page(s) 687 - 697
Ann. For. Sci. 59 (2002) 687-697
DOI: 10.1051/forest:2002057

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

Jean-François Trontin, Luc Harvengt, Elizabeth Garin, Manuel Lopez-Vernaza, Lydia Arancio, Josiane Hoebeke, Francis Canlet and Marc Pâques

AFOCEL, Laboratoire de Biotechnologie (, Domaine de l'Étançon, 77370 Nangis, France
(Received 1 September 2001; accepted 22 January 2002)

Using our improved protocols for somatic embryogenesis in Pinus pinaster, transgenic tissues and plantlets were recovered after microprojectile bombardment (biolistic) or cocultivation of embryonal-suspensor masses (ESM) with Agrobacterium tumefaciens. Transformation experiments were carried out with selectable hpt gene (hygromycin B resistance) and reporter gus gene ( $\beta$-glucuronidase activity). With both methods, hygromycin was shown to be an effective selective agent of transformed cells within 4-19 weeks. The mean number of hygromycin-resistant lines expressing gus per gram ESM subjected to DNA transfer, ranged from 7.0 to 8.5 using biolistic and 0 to 67.3 during Agrobacterium experiments. Mature somatic embryos obtained from some transformed lines were converted into plantlets and grown in the greenhouse. The whole process (from transformation to plant acclimatisation) could be completed within only 12 months. The transgenic state of ESM, somatic embryos and plants was confirmed by histochemical GUS assays and molecular methods.

Transformation génétique du pin maritime (Pinus pinaster Ait.). En appliquant nos protocoles d'embryogenèse somatique développés pour Pinus pinaster, des tissus et plantes transgéniques ont été obtenus après bombardement avec des microparticules (biolistique) ou coculture de masses embryonnaires (ESM) avec Agrobacterium tumefaciens . Les expériences de transformation ont été conduites à l'aide du gène de sélection hpt (résistance à l'hygromycine B) et du gène rapporteur gus (activité $\beta$-glucuronidase). L'hygromycine a permis de sélectionner efficacement les cellules transformées par ces deux méthodes en 4 à 19 semaines. Le nombre moyen de lignées résistantes à l'hygromycine exprimant le gène gus obtenu par gramme d'ESM varie de 7,0 à 8,5 (biolistique) ou de 0 à 67,3 (Agrobacterium). Les embryons matures obtenus à partir de certaines de ces lignées ont pu être convertis en plantules élevées en serre. Seulement 12 mois sont nécessaires de la transformation des ESM jusqu'à l'acclimatation des plantes. La nature transgénique des ESM, embryons somatiques et plantes, a été confirmée à l'aide de tests histochimiques " GUS " et de méthodes moléculaires.

Key words: Pinus pinaster / somatic embryogenesis / biolistic / Agrobacterium tumefaciens / transgenic plant

Mots clés : Pinus pinaster / embryogenèse somatique / biolistique / Agrobacterium tumefaciens / plante transgénique

Correspondence and reprints: Jean-François Trontin Tel.: +33 1 60670032; fax: +33 1 60670256;

© INRA, EDP Sciences 2002

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