The principal aim of this investigation was to improve somatic embryogenesis initiation and to enhance representation of families and genotypes within those families of Pinus radiata D. Don. A total of 19 open-pollinated seed families, many with unrelated and weakly related parents, were tested. Optimum stage of cone maturity for initiation success was tested by five collections made at 1 week intervals, spanning the developmental period from pro-embryo to cotyledonary embryos. Two media were compared; embryo-development media (EDM6) and a modified Litvay medium (Glitz). Two zygotic embryo explant-preparation techniques were tested; embryos with retained megagametophytes and excised embryos. Proliferating embryogenic tissues were obtained from all four treatments (2850 explants per treatment, 570 per collection time) for the 19 families. The best initiation rates were achieved with a combination of Glitz medium with excised zygotic embryos, with 55% of explants from all collections and all families combined giving rise to proliferating embryogenic tissue. At the optimal collection time for each of the families, this treatment gave a range of 47%–97% initiation success with an average of 70% per family.

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

This is the first published report comparing production and performance of adventitious shoots from cryopreserved cotyledons, with axillary shoots formed from epicotyls of the same zygotic embryo of radiata pine (Pinus radiata D. Don). Genotypes from 10 control-pollinated families of P. radiata in two treatments were compared for shoot initiation, in vitro growth, rooting, and early nursery performance. Plant growth in nursery beds was assessed by measuring height after 2 and 7 months. After 8 months in nursery beds, the physiological ages of genotypes were assessed before field planting. Genotype capture was higher from the cryopreserved cotyledons than from the epicotyls. This technique has the advantage of preserving juvenile material while field testing is done. Early shoot multiplication in both treatments was good. After four transfer cycles, epicotyl cultures showed improved elongation and higher multiplication rates. After 6 months of in vitro growth, shoots from both treatments were given auxin pulses. Shoots of adventitious origin were slower to root than epicotyl-derived shoots. Overall rooting rates were satisfactory. Plants of adventitious origin were shorter when planted into nursery beds and when transferred to field trials. Assessment of relative physiological age indicated that all plants of adventitious origin showed some increase.

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