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Shape change is a prevalent function apparent in a diverse set of natural structures, including seed dispersal units, climbing plants and carnivorous plants. Many of these natural materials change shape by using cellulose microfibrils at specific orientations to anisotropically restrict the swelling/shrinkage of their organic matrices upon external stimuli. This is in contrast to the material-specific mechanisms found in synthetic shape-memory systems. Here we propose a robust and universal method to replicate this unusual shape-changing mechanism of natural systems in artificial bioinspired composites. The technique is based upon the remote control of the orientation of reinforcing inorganic particles within the composite using a weak external magnetic field. Combining this reinforcement orientational control with swellable/shrinkable polymer matrices enables the creation of composites whose shape change can be programmed into the material’s microstructure rather than externally imposed. Such bioinspired approach can generate composites with unusual reversibility, twisting effects and site-specific programmable shape changes. Many living organisms use materials that are capable of changing their shape in response to changes in the environment. Erb et al . demonstrate an approach to synthesizing artificial shape-changing composites that replicate such behaviour.

Slash pine ( Pinus elliottii Engelm.) is an economically significant tree species valued for its timber and resin production. Establishing a somatic embryogenesis system is essential for the effective utilization of slash pine. This study aims to develop an optimized cultivation system for the long-term proliferation of embryogenic callus (EC) in slash pine. The study used the EC induced from immature zygotic embryos as materials to investigated the effects of plant growth regulators (PGRs), subculture cycles, and subculture durations on the EC proliferation coefficient and the number of somatic embryos (SE). Additionally, we monitored the changes in the proliferation coefficient, the number of SE, and physiological and biochemical indicators of EC during long-term proliferation. The EC proliferation coefficient reached its highest value (7.65) when the DCR medium was supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (6-BA). Refreshing the culture medium every 14 days, EC maintaining proliferation coefficient (4.05) and embryogenic potential for approximately three year (980 d), but the number of somatic embryos was low and significantly declined after 140 days.The additional supplementation of 1.0 mg/L 2- (1-naphthyl) acetic acid (NAA) to the original proliferation medium further enhanced SE induction, achieving a maximum of 143.25 SE per gram of EC. Under the optimal proliferation culture conditions, EC was subcultured for 392 days, with no significant differences observed in the proliferation coefficient and the number of SE. The results of correlation and path analyses between physiological indicators and proliferation coefficient indicate that the proliferation coefficient of EC is mainly related to the content of storage substances (starch and soluble sugars) and the activities of redox-related enzymes (POD and CAT). The optimized long-term proliferation culture system for EC in slash pine provides an efficient approach to enhance the production of EC and SE.

Background The strong genotype dependence of embryogenic callus (EC) induction limits the high-frequency regeneration of Korean pine ( Pinus koraiensis ) via somatic embryogenesis (SE). Methods We examined the morphological and histocytological features of various callus types using external morphology, microstructure, and ultrastructure analyses. To assess their embryogenic potential, we conducted somatic embryogenesis (SE) experiments. Additionally, we investigated programmed cell death (PCD) events associated with genotype-dependent embryogenic callus (EC) formation in Korean pine by performing TUNEL assays, detecting caspase-3 and vacuolar processing enzyme (VPE) activity, and measuring H 2 O 2 and O 2 •− levels. Results Obvious cell death was observed in the cell ultrastructure of LEC and NEC induced by recalcitrant genotypes. In LEC cells, we found typical characteristics of autolytic PCD, such as vacuole fusion, vacuolation of cells, invagination and rupture of the plasma membrane, few cytoplasm and organelles. In the NEC, degrading nucleus, withered cytoplasm and invagination and rupture of the plasma membrane were detected in early stage, but vacuole fusion was not observed. In addition, we observed DNA fragmentation into small fragments in LEC and NEC. Significant changes in the activities of caspase-3 and VPE, reactive oxygen species (ROS) levels, especially for H 2 O 2 , have been identified in LEC. However, in NEC, only the significant changes in the O 2 •− level and the caspase-3 activity were observed. Conclusion PCD was present in NEC and LEC cells in recalcitrant genotypes of Korean pine, which may be responsible for the loss or reduction of embryonic capacity of induced Korean pine callus. Our novel findings on PCD help elucidate the underlying causes of the difficulty in inducing EC from recalcitrant genotypes of Korean pine from a novel perspective.

Forests are considered to be an important pillar for a country’s bioeconomy but unfortunately are being depleted globally. Pinus roxburghii has considerable ecological and economic importance for Pakistan. It is successfully used as a pioneer species in afforestation and reforestation programs in the era of climate change. This paper reports on a significantly improved protocol for P. roxburghii somatic embryogenesis. Successive developmental stages for zygotic as well as somatic embryos were reported first time for this important pine species. Initiation of somatic embryogenesis was affected by the developmental stage of the zygotic embryo. Immature pre-cotyledonary zygotic embryos produced 33% initiation of embryonic masses on LP-889 medium supplemented with 11 µM Naphthaleneacetic acid (NAA), 2.5 µM Benzylaminopurine (BAP), 2.5 µM kinetin and 4 µM Abscisic acid (ABA). LP-1250 medium provided with 2 µM BAP, 2 µM kinetin, 5 µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 5 µM ABA was used for the proliferation and maintenance of embryogenic tissue. Polarized immature somatic embryos were clearly visible during the proliferation and maintenance phase. Two media; mLV and LP-1562 were tested as well as several combinations of L-glutamine and ABA for maturation. Maximum number of somatic embryos (310 embryos per g embryogenic tissue) was obtained on LP-1562 medium (containing 450 mg/l of L-glutamine and 20 µM ABA) as compared to 204 embryos per g embryogenic tissue on mLV medium (containing 900 mg/l L-glutamine and 60 µM ABA). Mature somatic embryos were formed on maturation medium after 6–8 weeks. Successful conversion of somatic embryos resulted after 12 weeks on LP-397 medium. However, low rooting rates (7% and 3%) were observed for somatic embryos formed on maturation media, i.e. LP-1562 and mLV, respectively and precocious germination of the mature somatic embryos still remains a serious constraint.

phenotype of plants derived from the cold epitype (characterized by a faster and higher proline increase, lower increases in ABA levels, no reduction in active cytokinins, and a better net photosynthesis rate recovery) could be attributed to low-temperature-induced epigenetic marks that were absent in the warm epitype.

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Background There are clear differences in embryo development between angiosperm and gymnosperm species. Most of the current knowledge on gene expression and regulation during plant embryo development has derived from studies on angiosperms species, in particular from the model plant Arabidopsis thaliana . The few published studies on transcript profiling of conifer embryogenesis show the existence of many putative embryo-specific transcripts without an assigned function. In order to extend the knowledge on the transcriptomic expression during conifer embryogenesis, we sequenced the transcriptome of zygotic embryos for several developmental stages that cover most of Pinus pinaster (maritime pine) embryogenesis. Results Total RNA samples collected from five zygotic embryo developmental stages were sequenced with Illumina technology. A de novo transcriptome was assembled as no genome sequence is yet published for Pinus pinaster . The transcriptome of reference for the period of zygotic embryogenesis in maritime pine contains 67,429 transcripts, which likely encode 58,527 proteins. The annotation shows a significant percentage, 31%, of predicted proteins exclusively present in pine embryogenesis. Functional categories and enrichment analysis of the differentially expressed transcripts evidenced carbohydrate transport and metabolism over-representation in early embryo stages, as highlighted by the identification of many putative glycoside hydrolases, possibly associated with cell wall modification, and carbohydrate transport transcripts. Moreover, the predominance of chromatin remodelling events was detected in early to middle embryogenesis, associated with an active synthesis of histones and their post-translational modifiers related to increased transcription, as well as silencing of transposons. Conclusions Our results extend the understanding of gene expression and regulation during zygotic embryogenesis in conifers and are a valuable resource to support further improvements in somatic embryogenesis for vegetative propagation of conifer species. Specific transcripts associated with carbohydrate metabolism, monosaccharide transport and epigenetic regulation seem to play an important role in pine early embryogenesis and may be a source of reliable molecular markers for early embryogenesis.

In this study, several improvements and simplifications of SE protocols in Pinus pinaster (Ait.), a species of economic importance in the regions of Western Europe, are described. These improvements pertained to all stages of SE including high initiation frequencies in eight control pollinated seed families, relatively high somatic embryo maturation yield when cells were coated with particles of activated charcoal and a rapid production of plants directly in a shade house. The SE initiation frequency from isolated zygotic embryos was high (up to 100%) and plants were produced from 11 embryogenic lines representing all crosses. Based on these results, the estimated number of somatic embryos required to produce 1,000 plants varied from slightly more than the required number of plants to more than double this number depending on the line. Such an estimate is critical in developing plant production strategy when a number of embryogenic lines are considered for production of clonal plants.

There are concerns that large-scale stand mortality due to mountain pine beetle (MPB) could greatly reduce natural regeneration of serotinous Rocky Mountain (RM) lodgepole pine ( Pinus contorta var. latifolia ) because the closed cones are held in place without the fire cue for cone opening. We selected 20 stands (five stands each of live [[control]], 3 years since MPB [[3-yr-MPB]], 6 years since MPB [[6-yr-MPB]], and 9 years since MPB [[9-yr-MPB]] mortality) in north central British Columbia, Canada. The goal was to determine partial loss of serotiny due to fall of crown-stored cones via breakage of branches and in situ opening of canopy cones throughout the 2008 and 2009 growing seasons. We also quantified seed release by the opening of forest-floor cones, loss of seed from rodent predation, and cone burial. Trees killed by MPB three years earlier dropped ∼∼3.5 times more cones via branch breakage compared to live stands. After six years, MPB-killed stands had released 45%% of their canopy seed bank through cone opening, cone fall due to breakage, and squirrel predation. Further losses of canopy seed banks are expected with time since we found 9-yr-MPB stands had 38%% more open canopy cones. This was countered by the development of a modest forest-floor seed bank (6%% of the original canopy seed bank) from burial of cones; this seed bank may be ecologically important if a fire or anthropogenic disturbance reexposes these cones. If adequate levels of regeneration are to occur, disturbances to create seedbeds must occur shortly after tree mortality, before the seed banks are lost. Our findings also suggest that the sustained seed rain (over at least nine years) after MPB outbreak may be beneficial for population growth of ground-foraging vertebrates. Our study adds insight to the seed ecology of serotinous pines under a potentially continental-wide insect outbreak, threatening vast forests adapted to regeneration after fire.

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.