Cathie Reeves

Somatic embryogenesis (SE) is an advanced vegetative propagation technology that, when used in combination with breeding and cryopreservation, offers the forest industry a powerful tool for the deployment of elite genotypes. Germination and acclimatization are critical and cost-intensive phases in the production of somatic plants. The efficient conversion of somatic embryos into robust plants is a necessity if a propagation protocol is to be successfully adopted by the industry. In this work, these late phases of the SE protocol of two pine species were investigated. A shortened germination protocol and more controlled acclimatization were investigated for Pinus radiata, testing embryos from 18 embryogenic cell lines. A more simplified protocol, including a cold storage phase, was also compared among 10 of these cell lines. A shortened germination period and more controlled protocols significantly improved the acclimatization of somatic embryos directly from the lab to the glasshouse. When results for all cell lines were pooled, there were significant improvements in all growth characteristics (shoot height, root length, root collar diameter, and root quadrant score). When the more simplified protocol involving cold storage was tested, improvements were seen in the root architecture. For Pinus sylvestris, the late phases of somatic embryogenesis were investigated on seven cell lines in a set of two trials (four to seven cell lines per trial). During the germination phase, a shortened and simplified in vitro period, a cold storage option and basal media were explored. Viable plants were obtained from all treatments. However, there is still the need to improve germination and related protocols together with growing regimes for Pinus sylvestris. The improvements to protocols presented here, particularly for Pinus radiata, result in greater survival and quality of somatic emblings, leading to reduced costs and increased confidence in the technology. Simplified protocols using a cold storage option show great promise and, with some further research, could lead to reductions in the cost of the technology.

Development of hybrid pines of Pinus radiata D. Don for commercial forestry presents an opportunity to diversify the current resource of plant material. Climate change and different land uses pose challenges, making alternative species necessary to guarantee wood and non-wood products in the future. Pinus radiata var. cedrosensis × Pinus attenuata hybrid possesses different attributes, such as tolerance to drought conditions, better growth and resistance to snow damage at higher altitudes, and more importantly, different wood quality characteristics. Embryogenic cell lines were successfully initiated reciprocal hybrids using as initial explants megagametophytes, excised zygotic embryos and excised zygotic embryos plus nurse culture. However, the questions raised were: does the initiation environment affect the conversion to somatic plantlets months later? Does the mother tree or the cross have an effect on the conversion to somatic plantlets? In the present work we analysed the maturation rate, number of somatic embryos, germination rate, and the ex-vitro growth in cell lines derived from different initiation treatments, mother tree species, and crosses. Differences were not observed for in vitro parameters such as maturation and germination. However, significant differences were observed due to the mother tree species in relation with the ex-vitro growth rates observed, being higher those in which P. radiata acted as a mother. Moreover, embryogenic cell lines from these hybrids were stored at −80 °C and regenerated after one and five years.

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.

Changes in the cellular content of three polyamines (putrescine, spermidine and spermine) were compared at different stages of development in zygotic and somatic embryos of Pinus radiata D. Don. During embryo development, both the zygotic and the somatic embryos showed a steady increase in spermidine content, with either a small decrease or no significant change in putrescine. This led to a several‐fold increase in spermidine/putrescine ratios during development of both types of embryos. Cell cultures of plant‐forming and non‐plant‐forming lines derived from the same clone and growing on proliferation (maintenance) medium differed significantly in their polyamine levels. Mature, cotyledonary stage somatic embryos capable of germination and formation of plants could be distinguished by their higher spermidine/putrescine ratios from abnormal cotyledonary stage somatic embryos which were incapable of forming plants.