Cryo-Treatment Enhances the Embryogenicity of Mature Somatic Embryos via the lncRNA-miRNA-mRNA Network in White Spruce.

In conifers, somatic embryogenesis is uniquely initiated from immature embryos in a narrow time window, which is considerably hindered by the difficulty to induce embryogenic tissue (ET) from other tissues, including mature somatic embryos. In this study, the embryogenic ability of newly induced ET and DNA methylation levels was detected, and whole-transcriptome sequencing analyses were carried out. The results showed that ultra-low temperature treatment significantly enhanced ET induction from mature somatic embryos, with the induction rate from 0.4% to 15.5%, but the underlying mechanisms remain unclear. The newly induced ET showed higher capability in generating mature embryos than the original ET. DNA methylation levels fluctuated during the ET induction process. Here, WGCNA analysis revealed that OPT4, TIP1-1, Chi I, GASA5, GST, LAX3, WRKY7, MYBS3, LRR-RLK, PBL7, and WIN1 genes are involved in stress response and auxin signal transduction. Through co-expression analysis, lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might bind to pre-novel_miR_339 to promote the expression of WRKY7 genes for stress response; LAX3 could be protected by lncRNAs MSTRG.1070680.1 and MSTRG.33602.1 via serving as sponges for novel_miR_495 to initiate auxin signal transduction; lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might serve as sponges for novel_miR_527 to enhance the expression of Chi I for early somatic embryo development. This study provides new insight into the area of stress-enhanced early somatic embryogenesis in conifers, which is also attributable to practical applications.

Somatic embryogenesis in mature zygotic embryos of Picea pungens.

This study developed somatic embryogenesis protocols for Picea pungens (Engelm), an important ornamental species, including initiation, proliferation, maturation, germination, and acclimation. Somatic embryogenic tissues were induced from mature zygotic embryos of five families, with a frequency of [Formula: see text] 22% for each. Embryogenic tissues (ET) from 13 clones of three families were proliferated for one week, achieving an average rate of 179.1%. The ET of 38 clones of three families were cultured in maturation medium for six weeks; 188 mature embryos on average were counted per gram ET cultured, of which [Formula: see text] 81.1% appeared normal, and each clone developed at least 28 normally matured embryos. A total of 69.9% or more of cotyledonary somatic embryos germinated normally and developed into normal emblings. The experiment of transplanting the emblings into a greenhouse had an average survival rate of 68.5%. Considerable variation among and within families during initiation and proliferation was observed, but this variation decreased in the maturation and germination. Changing the concentration of plant growth regulator of the initiation medium did not significantly change the initiation frequency. We recommend incorporating these protocols into the current Picea pungens practical programs, although further research is essential to increase efficiencies and reduce cost.

Different Roles of Auxins in Somatic Embryogenesis Efficiency in Two Picea Species.

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.

Regulation of Somatic Embryo Development in Norway Spruce.

Somatic embryogenesis in Norway spruce combined with reverse genetics can be used as a model to study the regulation of embryo development in conifers. The somatic embryo system includes a sequence of developmental stages, which are similar in morphology to their zygotic counterparts. The system can be sufficiently synchronized to enable the collection and study of a large number of somatic embryos at each developmental stage.Here we describe a protocol for establishing transgenic cell lines in which genes of interest are upregulated or downregulated. Furthermore, we present methods for comparing embryo morphology and development in transgenic and control cell lines, including phenotyping the embryos, histological analysis, and tracking embryo development. The expression pattern of different genes is determined by GUS reporter assays.

Tissue-specific transposon-associated small RNAs in the gymnosperm tree, Norway spruce.

BACKGROUND: Small RNAs (sRNAs) are regulatory molecules impacting on gene expression and transposon activity. MicroRNAs (miRNAs) are responsible for tissue-specific and environmentally-induced gene repression. Short interfering RNAs (siRNA) are constitutively involved in transposon silencing across different type of tissues. The male gametophyte in angiosperms has a unique set of sRNAs compared to vegetative tissues, including phased siRNAs from intergenic or genic regions, or epigenetically activated siRNAs. This is contrasted by a lack of knowledge about the sRNA profile of the male gametophyte of gymnosperms. RESULTS: Here, we isolated mature pollen from male cones of Norway spruce and investigated its sRNA profiles. While 21-nt sRNAs is the major size class of sRNAs in needles, in pollen 21-nt and 24-nt sRNAs are the most abundant size classes. Although the 24-nt sRNAs were exclusively derived from TEs in pollen, both 21-nt and 24-nt sRNAs were associated with TEs. We also investigated sRNAs from somatic embryonic callus, which has been reported to contain 24-nt sRNAs. Our data show that the 24-nt sRNA profiles are tissue-specific and differ between pollen and cell culture. CONCLUSION: Our data reveal that gymnosperm pollen, like angiosperm pollen, has a unique sRNA profile, differing from vegetative leaf tissue. Thus, our results reveal that angiosperm and gymnosperm pollen produce new size classes not present in vegetative tissues; while in angiosperm pollen 21-nt sRNAs are generated, in the gymnosperm Norway spruce 24-nt sRNAs are generated. The tissue-specific production of distinct TE-derived sRNAs in angiosperms and gymnosperms provides insights into the diversification process of sRNAs in TE silencing pathways between the two groups of seed plants.

Effects of copper and arsenic stress on the development of Norway spruce somatic embryos and their visualization with the environmental scanning electron microscope.

Somatic embryogenesis is an important biotechnological technique which can be used in studies associated with environmental stress. Four embryogenic cell lines of Norway spruce were grown on media enriched with copper and arsenic in concentration ranges 50-500 µM and 10-50 µM, respectively. The effects were observed during subsequent stages of somatic embryogenesis, the characteristics evaluated being proliferation potential, average number of somatic embryos obtained per g/fresh weight, morphology of developed somatic embryos, metal uptake, and microanalysis of macro- and micronutrients uptake. Copper and arsenic at higher concentrations significantly reduced the growth of early somatic embryos. In almost all treatments, the cell line V-1-3 showed the best performance compared with the other lines tested. Environmental scanning electron microscopy was used to visualize and identify morphological abnormalities in the development of somatic embryos. Abnormalities observed were classified into several categories: meristemless somatic embryos, somatic embryos with disrupted meristem, reduced number of cotyledons, single cotyledon and fused cotyledons. With the application of a low temperature method for the environmental scanning electron microscope, samples were stabilized and whole meristems could be investigated in their native state. As far as we are aware, this is the first report of the effect of copper and arsenic during the process of somatic embryogenesis and the first to evaluate the content of macro and micronutrients uptake in Norway spruce.

Metabolome and transcriptome profiling reveal new insights into somatic embryo germination in Norway spruce (Picea abies).

Transcriptome, metabolome and histological profiling were performed on normal and aberrant somatic embryo germinants of Norway spruce (Picea abies L. Karst) providing a simplistic systems biology description of conifer germination. Aberrant germinants (AGs) formed periderm-like tissue at the apical pole and lacked shoot growth above the cotyledons. Transcriptome profiling (RNA-Sequencing) revealed a total of 370 differentially expressed genes at ≥1 or ≤-1 log2-fold change, where 92% were down-regulated in AGs compared with normal germinants (NGs). Genes associated with shoot apical meristem formation were down-regulated in AGs, or not differentially expressed between AGs and NGs. Genes involved in hormone signaling and transport were also down-regulated. Metabolite profiling by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS revealed biochemical difference between AGs and NGs, notably increased levels of sugars including glucose in AGs. Genes involved in glucose signaling were down-regulated and genes involved in starch biosynthesis were up-regulated, suggesting involvement of sugar signaling during late embryo development and germination. The overall results provide new data enabling further studies to confirm potential markers for a normal germination process in conifers.

Gene expression analysis of primordial shoot explants collected from mature white spruce (Picea glauca) trees that differ in their responsiveness to somatic embryogenesis induction.

Within a plantation of clonal somatic embryo-derived white spruce trees that belonged to four genotypes, one genotype (G6) has consistently responded for the last 16 years, to the induction of somatic embryogenesis within primordial shoot explants. Analysis of fourteen individuals within this genotype subsequently revealed a group of clonal trees that were nonresponsive. This in turn provided a unique opportunity to conduct differential gene expression analysis in the absence of genotype-specific factors. Absolute qPCR was first used to expand the analysis of several genes previously identified via microarray analysis to be differentially expressed during SE induction, along with the inclusion of two nonresponsive genotypes. While this demonstrated a high level of repeatability within, and between, responsive and nonresponsive genotypes, it did not support our previous contention that an adaptive stress response plays a role in SE induction responsiveness, at least with respect to the candidate genes we analyzed. RNAseq analysis was then used to compare responsive and nonresponsive G6 primordial shoots during the somatic embryogenesis induction treatment. Although not analyzed in this study, this included samples of callus and embryonal masses previously generated from G6 explants. In addition to revealing a large number of differentially expressed genes, de novo assembly of unmapped reads was used to generate over 25,000 contigs that potentially represent previously unidentified transcripts. This included a MADS-domain gene that was found to be the most highly differentially expressed gene within responsive shoot explants during the first seven days of the induction treatment.

Nitrogen uptake and assimilation in proliferating embryogenic cultures of Norway spruce-Investigating the specific role of glutamine.

Somatic embryogenesis is an in vitro system employed for plant propagation and the study of embryo development. Nitrogen is essential for plant growth and development and, hence, the production of healthy embryos during somatic embryogenesis. Glutamine has been shown to increase plant biomass in many in vitro applications, including somatic embryogenesis. However, several aspects of nitrogen nutrition during somatic embryogenesis remain unclear. Therefore, we investigated the uptake and assimilation of nitrogen in Norway spruce pro-embryogenic masses to elucidate some of these aspects. In our study, addition of glutamine had a more positive effect on growth than inorganic nitrogen. The nitrogen uptake appeared to be regulated, with a strong preference for glutamine; 67% of the assimilated nitrogen in the free amino acid pool originated from glutamine-nitrogen. Glutamine addition also relieved the apparently limited metabolism (as evidenced by the low concentration of free amino acids) of pro-embryogenic masses grown on inorganic nitrogen only. The unusually high alanine concentration in the presence of glutamine, suggests that alanine biosynthesis was involved in alleviating these constraints. These findings inspire further studies of nitrogen nutrition during the somatic embryogenesis process; identifying the mechanism(s) that govern glutamine enhancement of pro-embryogenic masses growth is especially important in this regard.

Identification of novel miRNAs and miRNA expression profiling in embryogenic tissues of Picea balfouriana treated by 6-benzylaminopurine.

Here, we compared miRNA expression profiles in embryonic cell cultures of the conifer Picea balfouriana following application of the synthetic cytokinin 6-benzylaminopurine (6-BAP). We used next-generation sequencing to analyze three libraries of small RNAs from the treated embryogenic cell cultures and generated 24,000,000 raw reads from each of the libraries. Over 70 differentially regulated micro RNA (miRNA) families (≥2 fold change in expression) were identified between pairs of treatments. A quantitative analysis showed that miR3633 and miR1026 were upregulated in tissues with the highest embryogenic ability. These two miRNAs were predicted to target genes encoding receptor-like protein kinase and GAMYB transcription factors, respectively. In one library, miR1160, miR5638, miR1315, and miR5225 were downregulated. These four miRNAs were predicted to target genes encoding APETALA2, calmodulin-binding protein, and calcium-dependent protein kinase transcription factors. The expression patterns of the miRNAs and their targets were negatively correlated. Approximately 181 potentially novel P. balfouriana miRNAs were predicted from the three libraries, and seven were validated during the quantitative analysis. This study is the first report of differential miRNA regulation in tissues treated with 6-BAP during somatic embryogenesis. The differentially expressed miRNAs will be of value for investigating the mechanisms of embryogenic processes that are responsive to 6-BAP in P. balfouriana.

Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

BACKGROUND: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. RESULTS: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. CONCLUSIONS: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation.

Partial desiccation treatment (PDT) stimulates germination and enhances the conversion of conifer somatic embryos. To better understand the mechanisms underlying the responses of somatic embryos to PDT, we used proteomic and physiological analyses to investigate these responses during PDT in Picea asperata. Comparative proteomic analysis revealed that, during PDT, stress-related proteins were mainly involved in osmosis, endogenous hormones, antioxidative proteins, molecular chaperones and defence-related proteins. Compared with those in cotyledonary embryos before PDT, these stress-related proteins remained at high levels on days 7 (D7) and 14 (D14) of PDT. The proteins that differentially accumulated in the somatic embryos on D7 were mapped to stress and/or stimuli. They may also be involved in the glyoxylate cycle and the chitin metabolic process. The most significant difference in the differentially accumulated proteins occurred in the metabolic pathways of photosynthesis on D14. Furthermore, in accordance with the changes in stress-related proteins, analyses of changes in water content, abscisic acid, indoleacetic acid and H2 O2 levels in the embryos indicated that PDT is involved in water-deficit tolerance and affects endogenous hormones. Our results provide insight into the mechanisms responsible for the transition from morphologically mature to physiologically mature somatic embryos during the PDT process in P. asperata.

DNA methylome of the 20-gigabase Norway spruce genome.

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.

Imaging of Norway spruce early somatic embryos with the ESEM, Cryo-SEM and laser scanning microscope.

This article describes the surface structure of Norway spruce early somatic embryos (ESEs) as a typical culture with asynchronous development. The microstructure of extracellular matrix covering ESEs were observed using the environmental scanning electron microscope as a primary tool and using the scanning electron microscope with cryo attachment and laser electron microscope as a complementary tool allowing our results to be proven independently. The fresh samples were observed in conditions of the air environment of the environmental scanning electron microscope (ESEM) with the pressure from 550Pa to 690Pa and the low temperature of the sample from -18°C to -22°C. The samples were studied using two different types of detector to allow studying either the thin surface structure or material composition. The scanning electron microscope with cryo attachment was used for imaging frozen extracellular matrix microstructure with higher resolution. The combination of both electron microscopy methods was suitable for observation of "native" plant samples, allowing correct evaluation of our results, free of error and artifacts.

WUSCHEL-RELATED HOMEOBOX 2 is important for protoderm and suspensor development in the gymnosperm Norway spruce.

BACKGROUND: Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive. Here, we use somatic embryos and reverse genetics for studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce). RESULTS: The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny. PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor. Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm. Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass. In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was reduced. Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos. On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation. CONCLUSIONS: Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants. In addition, PaWOX2 might exert a unique function in suspensor expansion in gymnosperms.

Transcriptomic and proteomic analyses of embryogenic tissues in Picea balfouriana treated with 6-benzylaminopurine.

The cytokinin 6-benzylaminopurine (6-BAP) influences the embryogenic capacity of the tissues of Picea balfouriana during long subculture (after 3 months). Tissues that proliferate in 3.6 and 5 µM 6-BAP exhibit the highest and lowest embryogenic capacity, respectively, generating 113 ± 6 and 23 ± 3 mature embryos per 100 mg of tissue. In this study, a comparative transcriptomic and proteomic approach was applied to characterize the genes and proteins that are differentially expressed among tissues under the influence of different levels of 6-BAP. A total of 51 375 unigenes and 2617 proteins were obtained after quality filtering. There were 2770 transcripts for proteins found among these unigenes. Gene ontology (GO) analysis of the differentially expressed unigenes and proteins showed that they were involved in cell and binding activity and were enriched in ribosome and glutathione metabolism pathways. Ribosomal proteins, glutathione S-transferase proteins, germin-like proteins and calmodulin-independent protein kinases were up-regulated in the embryogenic tissues with the highest embryogenic ability (treated with 3.6 µM 6-BAP), which was validated via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and these proteins might serve as molecular markers of embryogenic ability. Data are available via Sequence Read Archive (SRA) and ProteomeXchange with identifier SRP042246 and PXD001022, respectively.

Wuschel-related homeobox 8/9 is important for proper embryo patterning in the gymnosperm Norway spruce.

Proper embryo development is crucial as that is when the primary body axes are established. In Arabidopsis, AtWOX8 and AtWOX9, members of the Wuschel-related homeobox (WOX) gene family, are critical for embryo development. In Norway spruce, PaWOX8/9, which is expressed in embryos, is the homologue of AtWOX8 and AtWOX9. In this work, it is shown that the transcript abundance of PaWOX8/9 is high during early and late embryogeny and that it decreases when the maturation phase starts. To address the function of PaWOX8/9 during embryo development, RNAi lines were established to down-regulate the transcript level of PaWOX8/9, using both constitutive and inducible promoters. Embryos in the PaWOX8/9 RNAi lines show an aberrant morphology caused by disturbed orientation of the cell division plane at the basal part of the embryonal mass during early and late embryogeny. In addition, the transcript level of several key cell-cycle-regulating genes, for example, PaE2FAB-like and PaCYCLIN B-like, are affected in the PaWOX8/9 RNAi lines. Taken together, our results suggest that PaWOX8/9 may perform an evolutionarily conserved function as a regulator of the establishment of the apical-basal embryo pattern.

A possible biochemical basis for fructose-induced inhibition of embryo development in Norway spruce (Picea abies).

Sugars play an important role in various physiological processes during plant growth and development; however, the developmental roles and regulatory functions of hexoses other than glucose are still largely unclear. Recent studies suggest that blocked embryo development in Norway spruce (Picea abies (L.) Karst) is associated with accumulation of fructose. In the present study, the potential biochemical regulatory mechanism of glucose and fructose was studied during development of somatic embryos of Norway spruce from pro-embryogenic masses to mature embryos. The changes in protein fluorescence, a marker of the Maillard reaction, were monitored in two cell lines of Norway spruce that were grown on media containing sucrose (control), glucose or fructose. Manual time-lapse photography showed that growth of embryogenic cultures on medium containing sucrose was characterized by normal development of mature embryos whereas the embryogenic cultures that were grown on media containing glucose or fructose did not develop mature embryos. The biochemical analyses of embryogenic samples collected during embryo development showed that: (i) the content of glucose and fructose in the embryogenic cultures increased significantly during growth on each medium, respectively; (ii) the accumulation of Maillard products in the embryogenic cultures was highly correlated with the endogenous content of fructose but not glucose; and (iii) the embryogenic cultures grown on fructose displayed the highest protein carbonyl content and DNA damage whereas the highest content of glutathione was recorded in the embryogenic cultures that had grown on sucrose. Our data suggest that blocked development of embryos in the presence of fructose may be associated with the Maillard reaction.

The anti-actin drugs latrunculin and cytochalasin affect the maturation of spruce somatic embryos in different ways.

The role of the actin cytoskeleton in somatic embryo development was investigated using latrunculin B and cytochalasin D. Brief treatments (1h) with either drug at the start of maturation fragmented the actin in suspensor cells and/or depolymerized actin filaments in meristematic cells. The drugs targeted different cells: latB primarily affected the suspensor cells, but cchD damaged both suspensor and meristematic cells. Lethal damage to the meristematic and suspensor cells was observed when the drugs were applied throughout the maturation period, although the severity of this effect depended on their concentrations. The drugs' effects on the yield of mature somatic embryos were investigated by applying them to embryo cultures throughout the maturation period or for one week at three different points in the maturation process: immediately prior to the start of maturation, during the first week of maturation, and during the fourth week of maturation. The strongest effects were observed when the drugs were applied at the start of maturation. Under these conditions, latB destroyed the suspensors, eliminating the underdeveloped embryos that depend on them. This accelerated the development of embryos that were capable of separating from the suspensors. Thus, while the total number of embryos at the end of the maturation period was lower than in untreated control cultures, the surviving mature embryos were of high quality. cchD treatment at the start of maturation strongly inhibited embryo development. Drug treatment at the end of the maturation period did not significantly affect embryo development: latB caused no change in the yield of somatic embryos, but cchD treatment increased the number of malformed embryos compared to untreated controls.