Cryopreservation of Pinus radiata zygotic embryo cotyledons: effect of storage duration on adventitious shoot formation and plant growth after 2 years in the field

Abstract

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

Genome fidelity during short- and long-term tissue culture and differentially cryostored meristems of silver birch (Betula pendula)

“…short-or long-term tissue culture or different cryopreservation protocols had no effect on the growth of the regenerated plants. In woody plants, the only report of the potential effect of cryopreservation on growth of regenerated plants is published by Hargreaves et al (2004). They found the height growth of radiata pine (Pinus radiata D. Don) similar both following cryopreservation and micropropagation.…”

A comparative evaluation of the application of somatic embryogenesis, rooting of cuttings, and organogenesis of conifers

“…It was found that partially desiccated cotyledons of P. radiata, detached from mature zygotic embryos, can be cryopreserved without cryoprotectant pretreatment with one-step cooling. After up to 28 days in cryopreservation, the thawed cotyledons produced the same number of adventitious shoots and plants as the noncryopreserved control cotyledons, but plant height was 8% lower in the cryopreserved material after 21 months in the field (Hargreaves et al 2004). Subsequently, it was found that more genotypes were captured by culture of cryopreserved cotyledons than were obtained from axillary shoots arising from epicotyls of the noncryopreserved parts of the same zygotic embryo (Hargreaves et al 2005).…”

“…This is not long enough for a proper field test. The ability to cryopreserve material for propagation by organogenesis is desired for species for which organogenesis provides plantlets for a large number of genotypes per family, whereas SE does so for only a limited number of genotypes within the family (Hargreaves et al 2004(Hargreaves et al , 2011. It was found that partially desiccated cotyledons of P. radiata, detached from mature zygotic embryos, can be cryopreserved without cryoprotectant pretreatment with one-step cooling.…”

Detection of asymptomatic fungal microorganisms in Pinus radiata tissue culture material

“…The adventitious-axillary organogenesis shoots were prepared from excised cotyledons that were cryopreserved at − 196°C and then had shoots initiated adventitiously. Non-germinated hypocotyls (including epicotyls) from the embryos that provided the cotyledons for cryopreservation were retained and these provided the noncryopreserved epicotyl-axillary shoots, thus material was available from two methodology origins using the same genotypes (Hargreaves et al 2004(Hargreaves et al , 2005. Mature material was established using techniques described by Horgan and Holland (1989).…”