Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in Norway spruce

Planta - A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and...     

Plant regeneration in Stone pine (<Emphasis Type="Italic">Pinus pinea</Emphasis> L.) by somatic embryogenesis

Regeneration of plants by somatic embryogenesis (SE) was achieved in Stone pine (Pinus pinea), one of the most characteristic tree species of the Mediterranean ecosystem. The initial explants were megagametophytes containing zygotic embryos from five selected half-sib families collected at different dates over 2 consecutive years. Rates of extrusion and initiation of SE differed in both years. However, qualitative patterns were very similar: for most families, the responsive developmental window was from late cleavage polyembryony to early cotyledonary stage. The highest overall mean frequencies of extrusion and SE initiation (7 and 0.9%, respectively, for the five families and the eight 2006 collections) were obtained on a modified Litvay’s medium with 9 μM 2,4-D and 4.5 μM BAP, supplemented with L-glutamine and casein hydrolysate. Families showed large differences in frequencies of SE initiation from year to year. Only seven embryogenic lines were induced in 2005, representing three of the five families tested, whereas 34 lines from all the families were obtained in 2006. Proliferation of embryonal masses (EM) was significantly improved when they were subcultured after dispersing in liquid medium and collected on filter paper disks, instead of being subcultured as small clumps. This effect showed a significant interaction with genotype. Several preconditioning treatments and culture media combinations were tested for embryo development and maturation. The high proliferation rate of EM hampered somatic embryo development. However, up to 42 mature embryos from different lines of three of the five families were obtained, 23 of them germinated and seven converted into somatic seedlings.     

Effect of different cryoprotectant procedures on the recovery and maturation ability of cryopreserved <Emphasis Type="Italic">Pinus pinea</Emphasis> embryogenic lines of different ages

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.