Early Somatic Embryos Research Articles

Genome-wide analysis of the GLP gene family and overexpression of GLP1-5–1 to promote lignin accumulation during early somatic embryo development in Dimocarpus longan

Longan (Dimocarpus longan Lour.) is an economically important subtropical fruit tree. Its fruit quality and yield are affected by embryo development. As a plant seed germination marker gene, the germin-like protein (GLP) gene plays an important role in embryo development. However, the mechanism underlying the role of the GLP gene in somatic embryos is still unclear. Therefore, we conducted genome-wide identification of the longan GLP (DlGLP) gene and preliminarily verified the function of DlGLP1-5–1. Thirty-five genes were identified as longan GLP genes and divided into 8 subfamilies. Based on transcriptome data and qRT‒PCR results, DlGLP genes exhibited the highest expression levels in the root, and the expression of most DlGLPs was upregulated during the early somatic embryogenesis (SE) in longan and responded to high temperature stress and 2,4-D treatment; eight DlGLP genes were upregulated under MeJA treatment, and four of them were downregulated under ABA treatment. Subcellular localization showed that DlGLP5-8–2 and DlGLP1-5–1 were located in the cytoplasm and extracellular stroma/chloroplast, respectively. Overexpression of DIGLP1-5–1 in the globular embryos (GEs) of longan promoted the accumulation of lignin and decreased the H2O2 content by regulating the activities of ROS-related enzymes. The results provide a reference for the functional analysis of DlGLPs and related research on improving lignin accumulation in the agricultural industry through genetic engineering.

Involvement of 5mC DNA demethylation <i>via</i> 5-aza-2'-deoxycytidine in regulating gene expression during early somatic embryo development in white spruce (<i>Picea glauca)</i>

Involvement of 5mC DNA demethylation <i>via</i> 5-aza-2'-deoxycytidine in regulating gene expression during early somatic embryo development in white spruce (<i>Picea glauca)</i>

Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.

Whole Genome Analysis of SLs Pathway Genes and Functional Characterization of DlSMXL6 in Longan Early Somatic Embryo Development.

Strigolactones (SLs), a new class of plant hormones, are implicated in the regulation of various biological processes. However, the related family members and functions are not identified in longan (Dimocarpus longan Lour.). In this study, 23 genes in the CCD, D27, and SMXL family were identified in the longan genome. The phylogenetic relationships, gene structure, conserved motifs, promoter elements, and transcription factor-binding site predictions were comprehensively analysed. The expression profiles indicated that these genes may play important roles in longan organ development and abiotic stress responses, especially during early somatic embryogenesis (SE). Furthermore, GR24 (synthetic SL analogue) and Tis108 (SL biosynthesis inhibitor) could affect longan early SE by regulating the levels of endogenous IAA (indole-3-acetic acid), JA (jasmonic acid), GA (gibberellin), and ABA (abscisic acid). Overexpression of SMXL6 resulted in inhibition of longan SE by regulating the synthesis of SLs, carotenoids, and IAA levels. This study establishes a foundation for further investigation of SL genes and provides novel insights into their biological functions.

Morphological and Physiological Indicators for Screening Cell Lines with High Potential for Somatic Embryo Maturation at an Early Stage of Somatic Embryogenesis in Pinus Koraiensis.

Many cell lines in the embryogenic callus cannot produce somatic embryos (SEs) even if they meet the optimal SE maturation culture conditions during conifer somatic embryogenesis. This phenomenon hinders the progress of the industrial-scale reproduction of conifers. Therefore, there is an urgent need to obtain morphological and physiological markers to screen embryogenic calli in response to SE maturation conditions. To detect cell lines with high somatic embryogenesis potential during the proliferation process, we counted the number of pro-embryos and early SEs (ESEs) in different cell lines and storage substances, endogenous hormones, and polyamine contents. The results showed that the yield of P. koraiensis SEs was heavily dependent on genotype (p = 0.001). There were high levels of PE III (pro-embryo III) number, ESE number, and soluble protein content, in the response cell lines (R cell lines), which were 1.6-, 3-, and 1.1-fold those of the obstructive cell lines (B cell lines), respectively. The B cell line had high levels of starch, auxin (IAA), Put, Spd, and putrescine: spermine (Put: Spm) compared to the R cell line. In addition, the numbers of PE III, ESEs, and soluble protein content were significantly positively correlated with SE yield. In contrast, the contents of starch, abscisic acid (ABA), Put, Spm, and Spd were significantly negatively correlated with SE yield. To ensure the accuracy of the results, we used nine cell lines to test the results. The PE III and ESE numbers and the Spm and Spd contents were positively correlated with SE yield, while the levels of starch, ABA, IAA, Put: Spd, and Put: Spm were negatively correlated with SE yield. Thus, we recommend using high PE III and ESEs as morphological indicators and low levels of starch, IAA, ABA, and Put: Spm as physiological markers to screen cell lines with a high somatic embryogenesis potential. In addition, we also found that the relationship between Spd, Spm, and SE yield was opposite in the two experimental results. Therefore, we speculate that the differences in Spd and Spm content are mainly affected by genotype. In conclusion, this study obtained the morphological and physiological markers of some high-somatic embryogenic cell lines by comparing the differences between nine somatic embryogenic cell lines. Our results can guide the improvement of conifer somatic embryogenesis technology and can provide a theoretical basis for accelerating the application of biotechnology in large-scale artificial breeding.

Morphological and endogenous phytohormone changes during long-term embryogenic cultures in Korean pine

In SE of Korean pine, decreasing embryogenic potential was observed in embryogenic callus (EC) with successive subcultures, especially when the cultures were maintained for more than 5–6 months. Thus, aiming to investigate the changes in morphology and the endogenous phytohormone associated with the decreasing somatic embryo differentiation capacity of Korean pine, here, we compared Korean pine calli with different embryogenic potential. It was found that both the external morphological characteristics and internal cell structure were significantly different among the different types of calli. Specifically, similar to zygotic counterparts of conifer trees, the calli with high embryogenic potential were characterized by a higher number of designated proembryogenic masses (PEMs) structure. In addition, surface of this types of callus was covered with a large amount of mucilaginous extracellular matrix (ECM), which could further differentiate into globular embryos containing the embryonal masses (EM) and embryonic tube (ET) structure.In contrast, in those low embryogenic potential calli, we observed disintegrated PEMs structure, a large amount of irregularly shaped cells, and degraded cells, which failed to form somatic embryos. The endogenous hormone results showed that high levels of abscisic acid (ABA) were beneficial for promoting early somatic embryo differentiation in Korean pine, but maintaining high embryogenic potential required moderate levels of indole-3-acetic acid (IAA), and low levels of zeatin-riboside (ZR). Additionally, a high ratio of IAA/ABA, methyl jasmonate (MeJA)/ABA, and gibberellins (GA3)/ABA were conducive to maintaining high embryogenic potential, whereas a low ratio of IAA/ABA and MeJA/ABA were effective for early somatic embryos morphological differentiation. The results can provide morphological and physiological indicators for identifying high embryogenic potential calli suitable for SE and help determine the best protocol on addition of exogenous hormones in the construction of a maintainable system of EC in Korean pine.

PAs Regulate Early Somatic Embryo Development by Changing the Gene Expression Level and the Hormonal Balance in Dimocarpus longan Lour.

Polyamines (PAs) play an important regulatory role in many basic cellular processes and physiological and biochemical processes. However, there are few studies on the identification of PA biosynthesis and metabolism family members and the role of PAs in the transition of plant embryogenic calli (EC) into globular embryos (GE), especially in perennial woody plants. We identified 20 genes involved in PA biosynthesis and metabolism from the third-generation genome of longan (Dimocarpus longan Lour.). There were no significant differences between longan and other species regarding the number of members, and they had high similarity with Citrus sinensis. Light, plant hormones and a variety of stress cis-acting elements were found in these family members. The biosynthesis and metabolism of PAs in longan were mainly completed by DlADC2, DlSAMDC2, DlSAMDC3, DlSPDS1A, DlSPMS, DlCuAOB, DlCuAO3A, DlPAO2 and DlPAO4B. In addition, 0.01 mmol∙L−1 1-aminocyclopropane-1-carboxylic acid (ACC), putrescine (Put) and spermine (Spm), could promote the transformation of EC into GE, and Spm treatment had the best effect, while 0.01 mmol∙L−1 D-arginine (D-arg) treatment inhibited the process. The period between the 9th and 11th days was key for the transformation of EC into GE in longan. There were higher levels of gibberellin (GA), salicylic acid (SA) and abscisic acid (ABA) and lower levels of indole-3-acetic acid (IAA), ethylene and hydrogen peroxide (H2O2) in this key period. The expression levels in this period of DlADC2, DlODC, DlSPDS1A, DlCuAOB and DlPAO4B were upregulated, while those of DlSAMDC2 and DlSPMS were downregulated. These results showed that the exogenous ACC, D-arg and PAs could regulate the transformation of EC into GE in longan by changing the content of endogenous hormones and the expression levels of PA biosynthesis and metabolism genes. This study provided a foundation for further determining the physicochemical properties and molecular evolution characteristics of the PA biosynthesis and metabolism gene families, and explored the mechanism of PAs and ethylene for regulating the transformation of plant EC into GE.

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.

Stage-specific protein regulation during somatic embryo development of Carica papaya L. ‘Golden’

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.

A peak in global DNA methylation is a key step to initiate the somatic embryogenesis of coconut palm (Cocos nucifera L).

DNA methylation, morphogenesis and gene expression during the somatic embryogenesis of Coconut are affected by 5-Azacytidine pretreatments, indicating that DNA methylation is an important factor throughout this process. Somatic embryogenesis (SE) is a process that can aid in the production of elite Cocos nucifera palms. It has been well established that epigenetic mechanisms are regulators of cell differentiation programs; however, their role in the coconut somatic embryogenesis has not yet been addressed. To this end, the morphogenetic changes, the global DNA methylation and the expression profiles of the SE-related genes and DNA methyltransferases genes were evaluated during the SE process, with and without the presence of 5-Azacytidine (AzaC). The results show that three days of pretreatments with 15µM and 20µM of AzaC significantly increased early somatic embryo formation (four- and tenfold, respectively). A clear peak of the global percentage of DNA methylation (approximately 13%) was determined at the beginning of the culture, followed by a re-establishing stage and a steady increase thereafter; in all cases, the levels of DNA methylation were lower after the pretreatments with AzaC. Additionally, the expression profiles of the SERK, WUS, BBM and LEC genes are modulated during the SE process and the pretreatments with AzaC affect the expression profiles of these genes, even at early stages. Furthermore, increased levels of expression were observed for the genes encoding for DNA methyltransferases (MET, CMT and DRM) at early and late stages of SE, indicating that DNA methylation is an important factor throughout the SE.

Physiological and structural responses of hybrid firs embryogenic tissue under cadmium stress

The study was focused on the investigation of cadmium (Cd) effect on the embryogenic tissue of hybrid firs growing under in vitro conditions. Two cell lines AC78 (Abies alba x A. cephalonica) and AN72 (Abies alba x A. numidica) were cultured on DCR medium supplemented with 100 and 250 µM of Cd(NO3)2·4H2O. The tissue accumulated heavy metal ions during the culture period and at the end of growth cycle 91.4% of Cd2+ present in the medium was accumulated in AN72 and 94.6% in cell line AC78. Higher concentration of Cd2+ affected negatively the growth of tissue exerted as decline in fresh as well as dry mass accumulation, although in comparison to control tissue statistically significant reduction has been recorded only for cell line AN72. The toxic effect of cadmium was also apparent on the micromorphology of early somatic embryos and resulted in loss of bipolarity as well as complete structural disintegration. The increased malondialdehyde content in response to Cd2+ in culture medium indicated lipid peroxidation as a consequence of membrane damage. The embryogenic tissue is suitable experimental system for the study of cadmium effect on the cells cultured in extremly controlled environment in vitro.

In-situ preparation of plant samples in ESEM for energy dispersive x-ray microanalysis and repetitive observation in SEM and ESEM

The Extended Low Temperature Method (ELTM) for the in-situ preparation of plant samples in an environmental scanning electron microscope enables carrying out repetitive topographical and material analysis at a higher resolution in the vacuum conditions of a scanning electron microscope or in the low gas pressure conditions of an environmental scanning electron microscope. The method does not require any chemical intervention and is thus suitable for imaging delicate structures rarely observable with common treatment methods. The method enables both sample stabilization as close to their native state as possible, as well as the transfer of the same sample from a low vacuum to an atmospheric condition for sample storage or later study. It is impossible for wet samples in the environmental scanning electron microscope. Our studies illustrate the high applicability of the ELTM for different types of plant tissue, from imaging of plant waxes at higher resolution, the morphological study of highly susceptible early somatic embryos to the elemental microanalysis of root cells. The method established here provides a very fast, universal and inexpensive solution for plant sample treatment usable in a commercial environmental scanning electron microscope equipped with a cooling Peltier stage.

Somatic Embryogenesis in Selected Conifer Trees Pinus nigra Arn. and Abies Hybrids.

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.

Somatic Embryogenesis and Plant Regeneration From Primordial Shoot Explants of Picea abies (L.) H. Karst. Somatic Trees.

The recalcitrance of adult conifer tissues has prevented vegetative propagation of trees with known and desired characteristics. Somatic embryogenesis (SE) initiation protocol, recently developed for white spruce (Picea glauca, Klimaszewska et al., 2011), was applied in order to examine the feasibility, frequency and timing of SE induction from primordial shoots (PS) of Norway spruce (P. abies). In total, 39 genotypes were screened from 2015 to 2017 using 4–6 years old trees of SE origin as explant donors. Two genotypes responded: 11Pa3794 produced six proliferating embryonal mass (EM) sublines and 11Pa4066 produced 23 EM sublines. SE initiations occurred at the beginning of April, when the temperature sum (d.d.) started to accumulate, and at the end of October or beginning of November when the chilling unit (ch.u.) sum was over 500. EM sublines from both genotypes contained numerous early somatic embryos as detected by acetocarmine staining. The sublines of 11Pa4066 produced the mean of 78.6 ± 12.8 cotyledonary somatic embryos /g FW, but 11Pa3794 produced only a few cotyledonary somatic embryos that were able to germinate. The original EM lines (from which the trees were regenerated) had produced the same number of somatic embryos in 2011 maturations, which was approximately 120 somatic embryos /g FW. Microsatellite analyses conducted with both responsive genotypes confirmed the genetic stability of the EM sublines compared with the donor trees growing in the field. SE protocol developed for white spruce PS explants was also suitable for PS of Norway spruce if the explants were in the responsive developmental stage.

Preparation of nitric oxide-releasing photo-crosslinked electrospun chitosan nanofibrous scaffolds for bone tissue engineering

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.

16S and 18S metagenomics analysis of fouling residues from aquaculture

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.

A novel energy-efficient version of the Calvin cycle

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.

Microbial electrochemical technology (MET) platform for turning carbon dioxide into a suitable substrate for a chain-elongation fermenter

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.

Improved and synchronized maturation of Norway spruce (Picea abies (L.) H.Karst.) somatic embryos in temporary immersion bioreactors

Somatic embryogenesis offers many benefits for clonal propagation in large-scale plant production of conifers. A key rate-limiting step is the conversion from early-stage somatic embryos in pro-embryogenic masses (PEMs) to the maturation stage. Immature embryos in PEMs are present at different developmental stages, where some are unable to respond to the maturation treatment, thus limiting yields of mature embryos. Synchronization of early somatic embryo development in PEMs could greatly improve subsequent yields of mature embryos. A temporary immersion bioreactor designed for Norway spruce (Picea abies (L.) H.Karst.) was used in this study. Through a specific system for dispersion, connected tissue of PEMs, composed of immature embryos grown in liquid medium in the temporary immersion bioreactors or on solid medium as a control, was dispersed and redistributed in a more uniform spatial arrangement. It was demonstrated that development of mature embryos could be significantly stimulated by dispersion, compared to controls, in both medium types. Synchronization of maturation was evaluated by a statistical approach. The present study shows that the yield of mature embryos from dispersed PEMs was three to five times higher than that from non-dispersed controls in three of four cell lines of Norway spruce tested, both in bioreactors and on solid medium.

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.