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Plant regeneration by somatic embryogenesis (SE) was achieved in Pinus pinea L., a forest species of economic importance for its edible seeds, but improvements in the SE protocol are needed to make this technology feasible for breeding programs. In the present study, different maturation treatments on medium with high concentration of sucrose and gelling agent were tested. The effects of abscisic acid (ABA) concentration and culture procedure, the presence of the auxin antagonist 2-(4-chlorophenoxy)2-methylpropionic acid (PCIB), and the partial desiccation of embryonal masses before maturation on the reduction of proliferation and promotion of maturation in six embryogenic lines were evaluated. Increasing ABA concentration neither reduced proliferation nor improved maturation. The highest number of mature embryos was produced with 121 μM ABA in line 1F11 or 161 μM ABA in line 2F47. The culture procedure did not affect growth rate, but monthly subcultures onto maturation medium increased the normal embryo production 13-fold by compared with no subculturing. PCIB decreased proliferation only when it was included during the 12 weeks of the maturation period, and did not improve somatic embryo production. Partial desiccation of embryonal masses between 5 to 26% water loss did not reduce proliferation but enhanced maturation by 1.7 to 4.7-fold compared with the control, depending on the embryogenic line. Up to 256 normal cotyledonary embryos per gram fresh weight from the best line and culture condition were obtained. Somatic embryos germinated and converted to plants at over 70%. Although improvements in maturation are provided, problems such as growth arrest of somatic seedlings and low rates of acclimatization still remain to be solved before SE can be used for large scale plant production in stone pine. [ABSTRACT FROM AUTHOR]

Strategies for genetic improvement programs of Pinus pinea L, an important tree species of the Mediterranean ecosystem, are focused on increasing pine nut yield. Somatic embryogenesis and cryopreservation of elite genotypes are emerging as key components of advanced forest breeding programs. This study was carried out with embryogenic lines of different ages obtained from selected half-sib families of the species. The effect of three cryoprotectant procedures on the recovery and maturation ability was tested in embryogenic lines that showed different growth rate, two of them at different ages. In general, cryopreservation drastically reduced growth rates of frozen and rewarmed tissues; however, the use of 5% PEG-sucrose-DMSO dramatically increased growth rates of rewarmed embryogenic cultures. Overall, embryogenic lines of stone pine were suitable for cryopreservation. Seven out of eight lines were recovered, although the initial growth rates were variable. Five of six lines including the three oldest ones were recovered using 5% PEG-sucrose-DMSO. No relation was observed between age and growth rate of embryogenic lines and their response to cryopreservation. The line 2F47 showed the most stable response after long-term subculture and recovery after cryopreservation, at different ages. On the contrary, younger embryogenic lines either recovered after cryopreservation or did not, depending on the applied procedure. Maturation of some of the older lines was restored or enhanced after cryopreservation. Somatic embryos were obtained in three out of five tested embryogenic lines recovered from cryopreservation. However, only a few plantlets from cryopreserved lines were regenerated indicating the process must be optimized further before it is a practical adjunct to breeding. [ABSTRACT FROM AUTHOR]

Main conclusion: Epigenetic memory affects the timing of bud burst phenology and the expression of bud burst-related genes in genetically identical Norway spruce epitypes in a manner usually associated with ecotypes. In Norway spruce, a temperature-dependent epigenetic memory established during embryogenesis affects the timing of bud burst and bud set in a reproducible and predictable manner. We hypothesize that the clinal variation in these phenological traits, which is associated with adaptation to growth under frost-free conditions, has an epigenetic component. In Norway spruce, dehydrins ( DHNs) have been associated with extreme frost tolerance. DHN transcript levels decrease gradually prior to flushing, a time when trees are highly sensitive to frost. Furthermore, EARLY BUD BREAK 1 genes ( EBB1) and the FT- TFL1- LIKE 2-gene ( PaFTL2) were previously suggested to be implied in control of bud phenology. Here we report an analysis of transcript levels of 12 DHNs, 3 EBB1 genes and FTL2 in epitypes of the same genotype generated at different epitype-inducing temperatures, before and during spring bud burst. Earlier flushing of epitypes originating from embryos developed at 18 °C as compared to 28 °C, was associated with differential expression of these genes between epitypes and between buds and last year's needles. The majority of these genes showed significantly different expressions between epitypes in at least one time point. The general trend in DHN expression pattern in buds showed the expected reduction in transcript levels when approaching flushing, whereas, surprisingly, transcript levels peaked later in needles, mainly at the moment of bud burst. Collectively, our results demonstrate that the epigenetic memory of temperature during embryogenesis affects bud burst phenology and expression of the bud burst-related DHN, EBB1 and FTL2 genes in genetically identical Norway spruce epitypes. [ABSTRACT FROM AUTHOR]

Main Conclusion: A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and histones, as well as sRNAs, are pivotal for the establishment of the epigenetic memory. As a forest tree species with long generation times, Norway spruce is remarkably well adapted to local environmental conditions despite having recently, from an evolutionary perspective, recolonized large areas following the last glaciation. In this species, there is an enigmatic epigenetic memory of the temperature conditions during embryogenesis that allows rapid adaptation to changing environment. We used a transcriptomic approach to investigate the molecular mechanisms underlying the formation of the epigenetic memory during somatic embryogenesis in Norway spruce. Nine mRNA libraries were prepared from three epitypes of the same genotype resulting from exposure to epitype-inducing temperatures of 18, 23 and 28 °C. RNA-Seq analysis revealed more than 10,000 differentially expressed genes (DEGs). The epitype-inducing conditions during SE were accompanied by marked transcriptomic changes for multiple gene models related to the epigenetic machinery. Out of 735 putative orthologs of epigenetic regulators, 329 were affected by the epitype-inducing temperatures and differentially expressed. The majority of DEGs among the epigenetic regulators was related to DNA and histone methylation, along with sRNA pathways and a range of putative thermosensing and signaling genes. These genes could be the main epigenetic regulators involved in formation of the epigenetic memory. We suggest considerable expansion of gene families of epigenetic regulators in Norway spruce compared to orthologous gene families in Populus and Arabidopsis. Obtained results provide a solid basis for further genome annotation and studies focusing on the importance of these candidate genes for the epigenetic memory formation. [ABSTRACT FROM AUTHOR]