Search

Xylem is required for the growth and development of higher plants to provide water and mineral elements. The thickening of the xylem secondary cell wall (SCW) not only improves plant survival, but also provides raw materials for industrial production. Numerous studies have found that transcription factors and non-coding RNAs regulate the process of SCW thickening. Pinus massoniana is an important woody tree species in China and is widely used to produce materials for construction, furniture, and packaging. However, the target genes of microRNAs (miRNAs) in the developing xylem of P. massoniana are not known. In this study, a total of 25 conserved miRNAs and 173 novel miRNAs were identified via small RNA sequencing, and 58 differentially expressed miRNAs were identified between the developing xylem (PM_X) and protoplasts isolated from the developing xylem (PM_XP); 26 of these miRNAs were significantly up-regulated in PM_XP compared with PM_X, and 32 were significantly down-regulated. A total of 153 target genes of 20 conserved miRNAs and 712 target genes of 113 novel miRNAs were verified by degradome sequencing. There may be conserved miRNA-mRNA modules (miRNA- MYB , miRNA- ARF , and miRNA- LAC ) involved in softwood and hardwood formation. The results of qRT-PCR-based parallel validation were in relatively high agreement. This study explored the potential regulatory network of miRNAs in the developing xylem of P. massoniana and provides new insights into wood formation in coniferous species.

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.

Vegetative propagation through somatic embryogenesis is critical in conifer biotechnology towards multivarietal forestry that uses elite varieties to cope with environmental and socio-economic issues. An important and still sub-optimal process during in vitro maturation of somatic embryos (SE) is the biosynthesis and deposition of storage proteins, which are rich in amino acids with high nitrogen (N) content, such as arginine. Mobilization of these N-rich proteins is essential for the germination and production of vigorous somatic seedlings. Somatic embryos accumulate lower levels of N reserves than zygotic embryos (ZE) at a similar stage of development. To understand the molecular basis for this difference, the arginine metabolic pathway has been characterized in maritime pine (Pinus pinaster Ait.). The genes involved in arginine metabolism have been identified and GFP-fusion constructs were used to locate the enzymes in different cellular compartments and clarify their metabolic roles during embryogenesis and germination. Analysis of gene expression during somatic embryo maturation revealed high levels of transcripts for genes involved in the biosynthesis and metabolic utilization of arginine. By contrast, enhanced expression levels were only observed during the last stages of maturation and germination of ZE, consistent with the adequate accumulation and mobilization of protein reserves. These results suggest that arginine metabolism is unbalanced in SE (simultaneous biosynthesis and degradation of arginine) and could explain the lower accumulation of storage proteins observed during the late stages of somatic embryogenesis.

The somatic embryogenesis in conifers represents a suitable model of plant regeneration system facilitating studies of fundamental aspects of an early development as well as in vitro micropropagation. The aim of our study was to deeper understand the somatic embryogenesis in the conifer tree Pinus nigra Arn. Comparative proteomic analysis based on 2D-PAGE in 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) initiated from cotyledons of somatic seedlings of the same genotypes, 3) embryogenic tissues that lost the maturation capacity (E-L) of two cell lines (E362, E366). Investigated pine tissues showed distinct structural features. The 24 protein spots were altered in both cell lines in comparison of embryogenic and non-embryogenic tissues. These proteins are involved in disease and defence mechanism, energy metabolism and biosynthesis of cell wall components. Two of three protein spots detected only in embryogenic form of both cell lines are similar to water deficit inducible protein LP3, the third remains uncharacterised. The loss of the maturation capacity was accompanied by changes in 35 and 38 protein spots in 362 and 366 cell lines, respectively. Only two of them were altered in both cell lines, suggesting non-uniform process of ageing., Biological Significance: Somatic embryogenesis in conifers represents an experimental system for the study of early plant development as well as a biotechnological tool for large-scale micropropagation. The obtained results give a new insight into the process of somatic embryogenesis of a conifer Pinus nigra Arn. by revealing differences at proteomic levels among in vitro cultured tissues characterised by different embryogenic potential. Microscopic investigations have also shown differences in the structural organisation of studied tissues., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Pinus radiata is an economically important conifer, and somatic embryogenesis is being currently used for its propagation. But the embryogenic competence of cultures decreases with culture age. To cope with this, cryopreservation protocols have been developed lately for different Pinus species. Although cryopreservation reduces the costs associated with embryogenic cultures maintenance, the initial investment and the maintenance of cryotanks are expensive when dealing with somatic embryogenesis basic research issues., Objective: To study the feasibility of storing embryogenic cell lines at -80 degree C for over a year., Materials and Methods: The feasibility of the conservation method was assessed in terms of recovery, maturation and germination rates., Results: The recovery rates were up to 77 percnt;, and maturation and germination rates were 86 percnt; and 83 percnt;, respectively., Conclusion: The work described here is a simple and low-cost protocol that enables successful conservation of embryogenic cell lines for over a year.

Zygotic embryos and megagametophytes of Pinus pumila for cultivation in vitro were transferred in 1/2 LV medium supplemented with growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (6-BAP) to induce somatic embryogenesis. Four stably proliferating cell lines from two genotypes were derived. The cell lines differed in the number of globular somatic embryos and the weight of embryogenic calli. Cells of these lines were multiplied as a result of somatic polyembryogenesis via cleavage. In the nutrient medium for maturation, mature somatic embryos were obtained. However, somatic embryos of not all embryogenic cell lines reached maturation. In this study, plantlets were obtained in an in vitro culture for the first time.

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.

Megagametophytes of Siberian pine were cultured on an in vitro culture medium 1/2 LV supplemented with growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (6-BAP) to form embryos. The competency of somatic cell of explants to embryogenesis manifested itself in an organized growth and polarity. A coenocyte consisting of long vacuolated cells was formed in the megagametophyte culture. Then, the migration of the nuclei to one of the poles of the cell, their division, and formation of embryoids was observed. The megagametophyte culture of the Siberian pine differed from the zygotic embryo culture by the absence of asymmetric division in the vacuolated cell.

Background: It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors., Results: Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in development, transcripts with homology to genes acting on modulation of auxin flow and determination of adaxial-abaxial polarity were up-regulated, as were putative orthologs of genes required for meristem formation and function as well as establishment of organ boundaries. Comparative analysis with A. thaliana embryogenesis also highlighted genes involved in auxin-mediated responses, as well as epigenetic regulation, indicating highly correlated transcript profiles between the two species., Conclusions: This is the first report of a time-course transcriptomic analysis of zygotic embryogenesis in a conifer. Taken together our results show that epigenetic regulation and transcriptional control related to auxin transport and response are critical during early to mid stages of pine embryogenesis and that important events during embryogenesis seem to be coordinated by putative orthologs of major developmental regulators in angiosperms.

Long-distance dispersal (LDD) of seeds has a critical impact on species survival in patchy landscapes. However, relative to pollen dispersal, empirical data on how seed LDD affects genetic diversity in fragmented populations have been poorly reported. Thus, we attempted to indirectly evaluate the influence of seed LDD by estimating maternal and paternal inbreeding in the seed rain of fragmented 8 Pinus densiflora populations. In total, the sample size was 458 seeds and 306 adult trees. Inbreeding was estimated by common parentage analysis to evaluate gene flow within populations and by sibship reconstruction analysis to estimate gene flow within and among populations. In the parentage analysis, the observed probability that sampled seeds had the same parents within populations was significantly larger than the expected probability in many populations. This result suggested that gene dispersal was limited to within populations. In the sibship reconstruction, many donors both within and among populations appeared to contribute to sampled seeds. Significant differences in sibling ratios were not detected between paternity and maternity. These results suggested that seed-mediated gene flow and pollen-mediated gene flow from outside population contributed some extent to high genetic diversity of the seed rain (H E > 0.854). We emphasize that pine seeds may have excellent potential for gene exchange within and among populations.

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.

Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150 degree C freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100 percent of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.

Understanding seed development is important for large-scale propagation and germplasm conservation for the Masson pine. We undertook a proteomic analysis of Masson pine seeds during the early stages of embryogenesis. Two-dimensional difference gel electrophoresis (2D DIGE) was used to quantify the differences in protein expression during early seed development. Using electrospray ionization mass spectrometry/mass spectrometry, we identified proteins from 43 gel spots that had been excised from preparative "pick" gels. Proteins involved in carbon metabolism were identified and were predominantly expressed at higher levels during the cleavage polyembryony and columnar embryo stages. Functional annotation of one seed protein revealed it involvement in programmed cell death and translation of selective mRNAs, which may play an important role in subordinate embryo elimination and suspensor degeneration in polyembryonic seed gymnosperms. Other identified proteins were associated with protein folding, nitrogen metabolism, disease/defense response, and protein storage, synthesis and stabilization. The comprehensive protein expression profiles generated by this study will provide new insights into the complex developmental process of seed development in Masson pine., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 µM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 µM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 µM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa., (© The Author 2011. Published by Oxford University Press. All rights reserved.)

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 approximately 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. Key words: biotic disturbance; cone burial; cone opening; Dendroctonus ponderosae; ground-foraging vertebrates; mountain pine beetle; natural regeneration; Pinus contorta var. latifolia; Rocky Mountain lodgepole pine; seed banks; serotiny (canopy seed storage); Tamiasciurus hudsonicus.

Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.

In plants, programmed cell death (PCD) is an important mechanism that controls normal growth and development as well as many defence responses. At present, research on PCD in different plant species is actively carried out due to the possibilities offered by modern methods in molecular biology and the increasing amount of genome data. The pine seed provides a favourable model for PCD because it represents an interesting inheritance of seed tissues as well as an anatomically well-described embryogenesis during which several tissues die via morphologically different PCD processes.

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P< or =0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.

Araucaria angustifolia is the only native conifer of economic importance in the Brazilian Atlantic Rainforest. Due to a clear-cutting form of exploitation this species has received the status of vulnerable. The aim of this work was to investigate and characterize changes in protein expression profile during seed development of this endangered species. For this, the proteome of developing seeds was characterized by 2-DE and LC-MS/MS. Ninety six proteins were confidently identified and classified according to their biological function and expression profile. Overaccumulated proteins in early seed development indicated a higher control on oxidative stress metabolism during this phase. In contrast, highly expressed proteins in late stages revealed an active metabolism, leading to carbon assimilation and storage compounds accumulation. Comprehensive protein expression profiles and identification of overaccumulated proteins provide new insights into the process of embryogenesis in this recalcitrant species. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed.

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.

OTUBAINS are a recently discovered family of cysteine proteases that participate in the ubiquitin pathway. These proteins were originally described in animal systems and are involved in removing the ubiquitin chain attached to a protein destined for degradation. In a cDNA-AFLP screen designed to identify genes that are expressed during early somatic embryogenesis in the conifer Pinus radiata, a fragment-derived transcript corresponding to an Otubain-like cysteine protease was identified. The full-length cDNA contained an 885 bp ORF encoding 294 amino acids, and was named PrOTUBAIN. The deduced protein showed high identity to other OTUBAINS and contained an OTU domain and a catalytic triad characteristic of cysteine proteases. The 3-D model of PrOTUBAIN showed significant similarity to human OTUBAIN2, suggesting that the plant protein may possess functions similar to that of the human protein. Real time PCR assays demonstrated that PrOTUBAIN is expressed in different tissues and that transcript are particularly abundant in embryogenic tissues. This is the first report of this class of protein in higher plants and the putative role of PrOTUBAIN is discussed.

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.

Analysis of cDNA-AFLPs was used to study gene expression underlying the early embryogenic process in the gymnosperm Pinus radiata. Somatic embryogenesis in this species was used as a model as it resulted in the generation of a large number of embryos at defined stages of development. The gene expression patterns of three embryogenic stages were compared with non-embryogenic cells. Fifty transcript-derived fragments (TDFs) that are upregulated and 32 TDFs that are down-regulated in the embryogenic stages were selected, sequenced and their homologies sought in the databases. Expression of a selected subset of differentially expressed genes was confirmed by RT-PCR and their levels of expression were quantified. Of the 50 up-regulated TDFs, 16 are homologous to genes encoding either known or putative proteins in higher plants, 19 are homologous to conifer ESTs and 15 did not show significant matches. Of the down-regulated TDFs, 8 are homologous to genes encoding either known or putative proteins, 20 are homologous to conifer ESTs and 4 of them did not show significant matches in DNA or protein sequence database. The known up-regulated genes were similar to genes involved in cellular metabolism and in the stress response and the known down-regulated genes were similar to genes involved in proteolysis, cell wall modification and signaling pathways. Their putative individual function is briefly reviewed based on published information, and the potential roles of these genes in embryo development are discussed.

Rab-related small GTP-binding proteins are known to be involved in the regulation of the vesicular transport system in eukaryotic cells. We report the characterization of a previously isolated full-length cDNA PpRab1 from Pinus pinaster. Amino acid sequence analysis revealed the presence of G1-G5 conserved domains of the GTPase Ras superfamily and a double cysteine motif in the C-terminal, characteristic of Rab proteins. The PpRab1 protein shows high sequence similarity to several Rab1 GTP-binding proteins in plants. Phylogenetic analysis showed that, within the Ras superfamily, PpRab1 is more closely related to the Rab family and within this, PpRab1 protein was found to cluster with Arabidopsis subfamily AtRABE, whose members are known to regulate ER-to-Golgi membrane trafficking steps. PpRab1 transcripts were expressed at constitutively high levels for the initial stages of zygotic embryo development, and then their relative abundance decreased as embryo matures. The PpRab1 transcript is not embryo-specific as it was found in roots, cotyledons and hypocotyls. An increase in PpRab1 expression level was observed when seeds are germinated and collected at successive time points of development. In situ RT-PCR analysis revealed an expression signal in early zygotic embryos. In view of the proposed roles of Rab1 GTP-binding protein, the possible function of the protein encoded by PpRab1 in embryogenesis is discussed.

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

The process of embryogenesis in gymnosperms differs in significant ways from the more widely studied process in angiosperms. To further our understanding of embryogenesis in gymnosperms, we have generated Expressed Sequence Tags (ESTs) from four cDNA libraries constructed from un-normalized, normalized, and subtracted RNA populations of zygotic and somatic embryos of loblolly pine (Pinus taeda L.). A total of 68,721 ESTs were generated from 68,131 cDNA clones. Following clustering and assembly, these sequences collapsed into 5,274 contigs and 6,880 singleton sequences for a total of 12,154 non-redundant sequences. Searches of a non-identical amino acid database revealed a putative homolog for 9,189 sequences, leaving 2,965 sequences with no known function. More extensive searches of additional plant sequence data sets revealed a putative homolog for all but 1,388 (11.4%) of the sequences. Using gene ontologies, a known function could be assigned for 5,495 of the 12,154 total non-redundant sequences with 13,633 associations in total assigned. When compared to approximately 72,000 sequences in a collated P. taeda transcript assembly derived from >245,000 ESTs derived from root, xylem, stem, needles, pollen cone, and shoot ESTs, 3,458 (28.5%) of the non-redundant embryo sequences were unique and thereby provide a valuable addition to development of a complete loblolly pine transcriptome. To assess similarities between angiosperm and gymnosperm embryo development, we examined our EST collection for putative homologs of angiosperm genes implicated in embryogenesis. Out of 108 angiosperm embryogenesis-related genes, homologs were present for 83 of these genes suggesting that pine contains similar genes for embryogenesis and that our RNA sampling methods were successful. We also identified sequences from the pine embryo transcriptome that have no known function and may contribute to the programming of gene expression and embryo development.

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.

Transmission electron microscopy of immunogold-labeled Chinese pine egg cells before and after fertilization revealed that polysomes are associated with microtubules (MTs) from fertilization to the 2-nucleate embryo stage. Ribosome aggregates of various size and shape were randomly distributed in the cytoplasm of the eggs before fertilization. Single MTs or clusters were observed to be free of polysomes at this stage. Upon fertilization, all polysomes were attached to MTs, and this association persisted until the formation of the polarized embryo. Thereafter, the polysomes spread into the cytoplasm and no polysome-MT association was observed in the embryo. Some of the polysomes were attached to one end of the MTs, while others appeared to form contacts along their entire length. No polysome-microfilament association was observed at any stage of the development. The polysome-MT association may provide a mechanism for MT-dependent mRNA localization in early embryo development of this plant.

Embryogenic cultures of loblolly pine (Pinus taeda L.), slash pine (Pinus elliottii Engelm.), longleaf pine (Pinus palustris Mill.) and slash pine x longleaf pine hybrids were initiated from immature seeds on an initiation medium containing 13.57 microM 2,4-dichlorophenoxyacetic acid and 2.22 microM benzylaminopurine. Embryogenic cultures proliferated and somatic embryos developed, matured and germinated following a modified protocol and media originally developed for radiata pine (Pinus radiata D. Don.) somatic seedling production. A discrete, light-sensitive pre-germination stage and a later germination (radicle emergence) stage were identified by the differential response of somatic embryos to light of different wavelengths. Different light quality treatments were applied during the pre-germination and germination steps, using cool white fluorescent bulbs or light-emitting diodes (LEDs), or both. In general, red wavelengths provided by LEDs during these steps resulted in higher frequencies of somatic embryo germination (up to 64%) and conversion (up to 50%), longer tap roots and more first-order lateral roots than the standard cool white fluorescent treatments or treatment with blue wavelengths from LEDs. In addition, exposure to red light allowed germination of somatic embryos of some clones that failed to produce germinants under fluorescent light. Germination and conversion were further enhanced by sequential application of cool white fluorescent light and red light, resulting in up to 100% germination and conversion in one experiment. Longleaf pine somatic embryos were especially responsive to the light quality treatments, resulting in the first report of somatic seedling production for this species.

A particle inflow gun was used to transfer the plasmid pAHC25 containing the bar gene conferring resistance to glufosinate and the gusA reporter gene, each driven by the maize ubiquitin promoter, to mature embryos of Pinus roxburghii (chir pine). High levels of transient expression were obtained when embryos were cultured for 6 days on 10 microM benzyl adenine-containing medium and then exposed to high osmoticum (0.5 M sucrose) before and after bombardment. Selection on medium containing Basta enabled recovery of stably transformed shoots, both from the epicotyl and from adventitious buds. The primary transformed shoots from the epicotyl were multiplied via axillary shoots. Transformation was confirmed by histochemical staining for beta-glucuronidase (GUS) activity, by polymerase chain reaction (PCR) amplification of fragments of gusA and nos terminator, and by the resistance of needles to Basta.

Here, embryo-specific patterns of glutamine synthetase (GS) genes were studied for the first time using pine somatic and zygotic embryogenesis as model systems. GS1a expression was absent in zygotic embryos whereas it was detected in the cotyledons of somatic embryos at late developmental stages along with transcripts for photosynthesis genes and arginase. These findings suggest that germination was initiated in maturing somatic embryos. GS1b transcripts were found mainly in procambial cells in both zygotic and somatic embryos. Expression of the GS1b in procambial cells before the differentiation of mature vascular elements indicated that this gene could be useful as a molecular marker for early stages of vascular differentiation in pine. Accordingly, a correlation was found between the quality of somatic embryos generated from three different cell lines and the pattern and level of GS1b expression. Our data suggest that GS1a and GS1b genes play distinct functional roles in the biosynthesis and mobilization of seed nitrogen reserves. Furthermore, the results presented may have potential application for improving conifer somatic embryogenesis.

In order to determine the suitability of reference or housekeeping genes as internal controls in real-time reverse transcriptase PCR (RT-PCR) assays for quantification of target mRNAs, we studied the levels of expression of four candidate reference genes in maritime pine by real-time RT-PCR. The expression levels obtained for glyceraldehyde-3-phosphate-dehydrogenase, 18S ribosomal RNA, eukaryotic translation initiation factor eIF4AII and ubiquitin in nine stages of embryo development revealed that none of the genes tested proved to be suitable as an internal control. Copy number quantification of the four transcripts showed an average relative variation of seven fold. We propose that the combination of a precise method for RNA quantification, internal controls for monitoring RT reaction and PCR efficiency and a robust external standard curve can guarantee a reliable absolute quantification of mRNA transcripts in real time RT-PCR. This approach may avoid the controversy in the use of housekeeping genes and may assume special significance in tissues undergoing developmental changes.

The stable transformation of embryogenic tissues of Pinus nigra Arn., cell line E104, has been achieved using a biolistic approach. The introduced DNA consisted of the uidA reporter gene under the control of the double CaMV 35S promoter and the nptII selection gene controlled by the single CaMV 35S promoter. Three days after bombardment, putative transformed tissues were selected for continued proliferation on a medium containing 20 mg geneticin l(-1). Resistant embryogenic tissue recovery required 10-12 weeks. The integration of the nptII and uidA genes was confirmed by both histochemical/fluorimetric GUS assays and PCR amplification of the inserts in the five geneticin resistant sub-lines of line E104. The activity of the uidA reporter gene in transgenic, embryogenic tissue lines was stable and could be detected after one year of culture. Somatic embryo maturation was, however, poor and no plantlet regeneration could be obtained.

A biolistic transformation procedure was used to transform embryogenic Pinus radiata tissue with constructs containing the Zea mays UBI1 (ubiquitin)-promoter followed by the P. radiata CAD (cinnamyl alcohol dehydrogenase) cDNA in sense or anti-sense orientation or in the form of an inverted-repeat. The effect of the different constructs on silencing the endogenous CAD gene was monitored in embryogenic tissue and somatic seedlings of 28 P. radiata transclones. Quantitative CAD measurements demonstrated that the construct containing an inverted-repeat of the CAD cDNA was most efficient in triggering gene silencing in P. radiata. Northern hybridization experiments with silenced transclones revealed that reduced CAD activities were the result of reduced steady state levels of the targeted CAD mRNA. Monitoring of the activity of the UBI1-promoter in the P. radiata transclones and heat-shock experiments with transgenic somatic P. radiata seedlings indicated that gene silencing is positively correlated with the expression level of the transgene. The obtained data are also consistent with a role for the expression level of the endogenous CAD gene in gene silencing.

In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles--plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine.

An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata. Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure. Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse. Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis. As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3. Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5. Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years. Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died. Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.

Clonal production of loblolly pine ( Pinus taeda L.) through somatic embryogenesis has the potential to meet the increasing industrial demands for high-quality uniform raw materials. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Twenty-five newly initiated loblolly pine genotypes were followed through the process of liquid culture establishment, embryo maturation, germination, and retrieval from cryogenic storage. A maturation medium, capable of promoting the development of loblolly pine somatic embryos that can germinate, is presented that combines 1/2 P6 modified salts, 2% maltose, 13% polyethylene glycol 8000 (PEG), 5 mg/l abscisic acid (ABA), and 2.5 g/l Gelrite. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also described. A set of somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed. The quality of the resulting embryos was examined and compared to that of zygotic embryos using such parameters as morphology, dry weight, germination performance, and gene expression. All of the observations that were made support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth.

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.

The PtNIP1;1 gene encodes an aquaglyceroporin that is expressed early in embryogenesis and appears to be expressed preferentially in the suspensor [V.T. Ciavatta et al. (2001) Plant Physiol 127:211-224]. An 899-bp fragment 5' to the PtNIP1;1 open reading frame (NIP(-899)) was cloned from loblolly pine (Pinus taeda L.) genomic DNA and fused to the beta-glucuronidase (GUS) reporter gene. The resulting plasmid, pNIP-GUS, was transformed into Norway spruce (Picea abies L.) embryogenic cultures by co-bombarding with a plasmid containing a bar gene construct as a selectable marker. The identity of lines selected on medium containing the herbicide Basta and showing beta-glucuronidase activity was confirmed by polymerase chain reaction as harboring GUS. Histochemical GUS assays of these lines revealed GUS activity in all cells of proembryogenic masses. During early embryogeny, GUS staining was intense in the suspensor region but not detectable in embryonal masses. GUS staining was absent by mid-embryogeny. By contrast, a control transgenic line, transformed with EuCAD-GUS, expressed GUS throughout embryo development. These results suggest that NIP(-899) contains elements that drive early embryogenesis-specific expression and suspensor-specific expression. This is the first example of a suspensor-specific promoter in conifers.

Early embryogeny of Pinus silvestris L. is characterized by suspensory system formation consisting of several cell generation formed by apical cell fissions. A comparative analysis was made of quantitative characteristics of nucleoli of two functionally different cell types: the apical cells of meristematic nature, and specialized cells resulting from mitotic cycle of suspensory cells. We determined the number of nucleoli and their diameters, the character of distribution and variability of these indices for cells of different types. It has been shown that in the cell cycle, changes of qualitative parameters of nucleoli in cells of secondary suspensors are most dynamic. A conclusion is made that in P. sylvestris L. early embryogeny differences in nucleolar activity and mechanisms of its regulation occur in suspensory cells of different generations. On the basis of differences in nucleolar activity observed in suspensory cells of the same generation, certain mechanisms of competition between cleavage polyembryos of this species are suggested.

The reproductive development of Pinus sylvestris from generative primordia to sporophyte initial (embryo tier of 16-celled proembryo) was studied. In the reproductive cycle, three types of stages are distinguished and described in association with: 1) hereditary program formation; 2) a new reproductive initial origin, and 3) increase in propogative energy. The layout of disposition of these stages in the reproductive cycle is given.

The comparative analysis of the genetic variation in the plantations of Pinus pallasiana D. Don in Krivbass and the Crimean populations has been conducted using the electrophoretic division of isoenzymes of 10 geneenzymous systems, encoded by 23 loci. In the artificial plantations of industrial ecotopes of Krivbass the level of genetic variation has been higher than in natural stands of Pinus pallasiana D. Don. within the range of one microslope in the Mountainous Crimea. The Nei' s genetic distance (Dn) between two regions varied within 0.016 to 0.026.