Abstract
Key messageThis paper describes the micropropagation of Douglas-fir via somatic embryogenesis using media and methods available in the public domain.Douglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programmes is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation is essential biotechnology tool in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on the modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Plant Cell Rep 4(6):325–328, 1985***). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realize increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France.
Highlights
Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) is the most important species in Pacific Northwest forests of the US and Canada, where it is native
Methods for somatic embryogenesis of Douglas-fir have been under development for several decades, with the first report from Durzan and Gupta in 1987 reporting initiation rates of up to 20–25% at the best collection date, obtained from immature zygotic embryos (ZEs) at the precotyledonary stage
Three years of initiation experiments have culminated in a treatment giving initiation rates ranging between 60.6 and 79.6% in five OP families (2012–2013 season)
Summary
Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) is the most important species in Pacific Northwest forests of the US and Canada, where it is native. In New Zealand, Douglas-fir accounts for approximately 6% (over 107,900 ha) of the sustainably managed exotic plantation forests, second only to Pinus radiata Douglas-fir US provenance/progeny collections were initiated in New Zealand in the 1950s and ranged from California to Washington (Shelbourne et al 2007; Dungey et al 2012; Low et al 2012). Douglas-fir produces abundant seed crops, these are at irregular intervals, from 2 to 10 years apart It is unlikely that significant quantities of controlled-pollinated seed will be available to supply New Zealand’s seed requirement for this species for a few years to come
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