New Approaches to Optimize Somatic Embryogenesis in Maritime Pine

Abstract

Maritime pine ( Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availability during SE process on the production of plants. A strong maternal effect on the embryogenic potential of maritime pine megagametophytes was observed in our experiments using half-sib and full-sib progenies, while paternal effect was almost undetectable. Besides, it seems possible to improve somatic embryo production of maritime pine megagametophytes by adjusting optimal temperature throughout the process: 28°C during induction and proliferation, and 23°C during the maturation phase. Using induction and proliferation media with reduced water availability (6 g/L Gelrite) can also increase embryo production. Since other limitation of maritime pine SE is culture decline of embryogenic masses (EMs), that reduces embryo yield and germination, we assessed the profile of ABA and IAA and the expression of two embryogenesis-related genes ( LEC1 and WOX2 ) during maturation of EMs of two morphotypes that differed in their maturation capability. Spiky morphotype (SK), with high maturation capability, had a steady increase in both hormones along the 12 weeks of the maturation, whereas ABA content in smooth morphotype picked at the 4th week and dropped. EMs with this morphotype also had a higher IAA content at the beginning of the maturation. A decrease of LEC1 and WOX2 gene expression over the course of embryo development was found to be characteristic of the SK with high maturation capability.

Proteome-Wide Analysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a Combined Response of Sugar Metabolism and Translational Regulation Mechanisms

“…Although not significant, the number of Se’s increased under high temperatures, a long-observed trend in our laboratory for both P. radiata and P. halepensis EMs initiated under high temperatures ( García-Mendiguren et al, 2016b ; Castander-Olarieta et al, 2019 ; Pereira et al, 2020 ). The same pattern was detected in similar temperature-based experiments during initiation of Pinus pinaster SE ( Arrillaga et al, 2019 ), confirming the role of temperature as an activator of embryo development. On top of that, Castander-Olarieta et al (2019) reported altered morphologies in both EMs and Se’s originating from different temperature treatments, and despite not observing germination and acclimatization differences, the resulting somatic plants showed changes at phenotypic level in terms of growth and stress-resilience ( Castander-Olarieta et al, 2020a ).…”

Barley somatic embryogenesis-an attempt to modify variation induced in tissue culture

“…The metAFLP is a choice for studies involving different aspects of tissue cultures. One such issue is the optimisation of plant regeneration via somatic embryogenesis [ 49 , 50 ], which could be performed if an adequate statistical approach is used to simultaneously cope with many factors and minimize experimental efforts [ 51 ]. The Taguchi approach [ 52 ] of experimental design optimisation could be considered for such purposes.…”

Priming Maritime Pine Megagametophytes during Somatic Embryogenesis Improved Plant Adaptation to Heat Stress

“…SE, the inducible process of obtaining multiple embryos from vegetative (somatic) cells, is the most promising vegetative propagation technology for conifers worldwide [25,26], since these species typically lack the capacity to be efficiently propagated using low-cost techniques (e.g., cuttings). SE protocols have been developed by our group [27,28] for maritime pine (Pinus pinaster Aiton), a species widely used for afforestation in the North-western Mediterranean area due to its fast growth, and the quality of its timber and oleoresins, as well as to its high phenotypic plasticity and stress tolerance. Furthermore, in Spain large genetic variation in adaptive traits have been found between maritime pine provenances [29,30].…”