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An efficient Biolistic® transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

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The present study was conducted to improve the transition from proliferation to maturation in embryogenic cultures of Nordmanns fir. For that reason, chemicals reported to affect endogenous levels or activity of auxin were included in the growth media during maturation. The auxin antagonist PCIB reduced proliferation and promoted the development of numerous high-quality mature embryos in the tested cell lines. PCIB could not substitute for exogenously supplied ABA and the positive effect was only found when PCIB and ABA were used in combination. The effect of PCIB was dependent on the concentration and the application period. The auxin transport inhibitor TIBA also reduced proliferation, but had no positive effect on maturation. The auxin synergist phloroglucinol had the opposite effect of PCIB; proliferation was increased and no maturation was initiated. A lowered concentration of boron had no effect on proliferation but had some positive effect on maturation. The optimum protocol for PCIB application was strongly genotype dependent, and a general scheme that covered the tested cell lines could not be found. Overexposure to PCIB during maturation caused abnormal development of the mature embryos, which was revealed by a reduced number of cotyledons. These results suggest that endogenously produced auxin may be one reason for low or failing maturation of embryogenic cultures of Nordmanns fir, but also imply that auxin may play a critical role for proper development of cotyledons during the later stages of embryo maturation.

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The biolistic® particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Several experiments with varying parameters were performed to increase the delivery efficiency. Six different controlling elements were cloned upstream of the ß-glucuronidase coding sequence (gusA reporter gene) and transient expression of the gusA reporter gene was compared three days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhancer-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-sequence) > CaMV 35S (without Kozak consensus sequence). Time course experiments monitoring gusA expression showed a significant decrease in the number of blue spots 10-14 days after bombardment. A few blue clumps however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic protocol for stable integration of genes into Pinus radiata embryogenic cultures which are capable of plant regeneration.

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This work is the first report of the cryopreservation of conifer cotyledons without cryoprotectants and their subsequent shoot regeneration and successful establishment of a field trial. Multiple genotypes of radiata pine (Pinus radiata D. Don) embryo cotyledons were stored in liquid nitrogen following a desiccation treatment. Cotyledons that had been stored in liquid nitrogen for 7, 14, and 28 days were compared with noncryopreserved cotyledons for adventitious shoot production, root formation on the shoots, and plant growth after 2 years in the field. Of the 72 genotypes tested, 79%-87% of them produced shoots on at least one treatment and 59% of them produced shoots on all treatments. Rooting rates of shoots were not affected by treatment, with the cryopreserved treatments rooting as well as the noncryopreserved controls. Height growth of plants in the nursery was similar across all treatments after 2 years but was influenced by setting date. The higher genotype capture possible with adventitious methodologies, compared with that of somatic embryogenesis, and the preservation of juvenile characteristics while material is stored in liquid nitrogen make adventitious methods worthy of intensive study for possible commercial application.Résumé : Cet article est le premier à rapporter la cryoconservation de cotylédons de conifères sans l'utilisation de cryoprotecteurs, la régénération subséquente de pousses et la réussite de leur établissement dans un test au champ. Plusieurs génotypes d'embryons de cotylédons de Pinus radiata D. Don ont été entreposés dans l'azote liquide après un traitement de dessiccation. Les cotylédons qui avaient été entreposés dans l'azote liquide pendant 7, 14 et 28 jours ont été comparés à des cotylédons non cryoconservés pour la production de pousses adventives, la formation de racines sur les pousses et la croissance des plants après deux ans au champ. Des 72 génotypes testés, 79-87 % de ceux-ci ont produit des pousses dans au moins un traitement et 59 % dans tous les traitements. Le taux d'enracinement des pousses n'était pas affecté par les traitements; l'enracinement était le même que les tissus aient été cryoconservés ou non. La croissance en hauteur des plants en pépinière était similaire dans tous les traitements après deux ans mais était influencée par la date de mise en terre. La conservation de plus de génotypes qui est rendue possible grâce aux métho-des utilisant des tissus adventifs, comparativement à l'embryogenèse somatique, et la préservation des caractères juvéniles pendant que le matériel est conservé dans l'azote liquide justifient l'étude plus poussée de ces méthodes qui pourraient avoir une application commerciale.[Traduit par la Rédaction] Hargreaves et al. 608