Somatic Embryo Development Research Articles

Functional analysis of the eTM-miR171-SCL6 module regulating somatic embryogenesis in Lilium pumilum DC. Fisch.

Somatic embryogenesis (SE) is of great significance in Lilium bulb production, germplasm preservation, and genetic improvement. miRNAs are important regulators of plant growth and development at the transcriptional level. Previous research by our group has shown that lpu-miR171 and its target gene SCARECROW-LIKE 6 (SCL6) play an important regulatory role in lily SE, and we predicted and identified that endogenous target mimics (eTMs) can regulate lpu-miR171. However, the associated mechanism and internal regulatory network are not yet clear. In the present study, lpu-miR171 was used as an entry point to explore the regulatory network between its upstream eTMs and its downstream target gene LpSCL6, as well as to identify the mechanism of this regulatory network in Lilium SE. Tobacco transient transformation confirmed that miRNA171 significantly inhibited the expression of LpSCL6. On this basis, the Lilium stable genetic transformation system was used to demonstrate that silencing lpu-miR171a and lpu-miR171b and overexpressing LpSCL6-II and LpSCL6-I promoted starch accumulation in calli and the expression of key cell cycle genes, thus providing energy to meet preconditions for SE and accelerate the formation and development of Lilium somatic embryos. LpSCL6-II and LpSCL6-I are nuclear proteins with self-activation activity in yeast cells. In addition, we confirmed in Lilium that lpu-eTM171 is the eTM of lpu-miR171 that binds lpu-miR171 to prevent cleavage of the target gene LpSCL6, thereby promoting SE. Therefore, the present study established a new mechanism whereby the eTM-miR171-SCL6 module regulates SE in Lilium pumilum and provides new insights clarifying the mechanism of SE.

Embryogenic cultures and somatic embryos development from mature seeds of jabuticaba (Plinia cauliflora (Mart.) Kausel)

Plinia cauliflora is an important Brazilian species that produces highly appreciated fruits, with a great potential of commercialization. However, the high cost of seedlings is a bottleneck for the expansion of commercial orchards. The present study aimed to investigate somatic embryogenesis as a propagation method for P. cauliflora using seeds as explants. To induce embryogenic mass (EM) and somatic embryo (SE) development we evaluated the supplementation of culture medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), combined or not with activated charcoal (AC). For the embryo maturation, we investigated the effects of AC, polyethylene glycol (PEG), Gelzan®, 6-benzylaminopurine and gibberellin supplementation. For the EM induction, the best results were obtained in MS culture medium supplemented with 300 μM 2,4-D and 1 g L-1 AC. During the first maturation phase, the supplementation of 30 g L-1 PEG improved the somatic embryo formation at the torpedo and cotyledonary stages, whereas the maturation treatments did not result in the conversion of the embryos into plantlets. The anatomical analysis showed that the 2,4-D presence for 60 days may have been deleterious for embryonic development. These results represent the first report of P. cauliflora somatic embryogenesis and its feasibility for mass propagation.

Expression analysis of OsSERK, OsLEC1 and OsWOX4 genes in rice (Oryza sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.

Inorganic Compounds that Aid in Obtaining Somatic Embryos.

Somatic embryogenesis (SE) is a process that allows formation of embryos from somatic cells; this biological process has different stages that first require micropropagation and conditioning of explant, and then induction, multiplication, development, and germination of somatic embryos (SoE), to obtain seedlings that will be acclimatized and grown in a greenhouse to further be cultivated in the field. Inorganic compounds are supplemented by macro- and micronutrients that can conform different culture media, and with other compounds such as a carbon source, vitamins, and plant growth regulators (PGRs), will direct the fate of the plant cells to obtain SoE that will regenerate into plants. The concentration of these inorganic compounds must be optimized, since at very high concentrations they can cause toxicity and at low concentrations they may not induce the desired response. The objective of this chapter is to describe the most significant advances in the use of inorganic elements during the different stages of SE, starting with the description of the most used basal media and later describing the use of the main studied mineral elements during establishment of SE.

Mini review: Application of the somatic embryogenesis technique in conifer species

The somatic embryogenesis (SE) process is better suited to large-scale production and automation than other clonal propagation methods such as the rooting of cuttings. SE is becoming a key technique to promote the asexual industrialization of conifers. Furthermore, somatic embryos are an ideal material to study the molecular mechanism of conifer embryo development, as the processes of somatic and zygotic embryo development are very similar. This brief review introduces the culturing techniques of the SE process in conifers and outlines the progress and deficiencies in conifer SE research. Emphasis is placed on the patterning formation of conifer somatic embryos.

Histological studies on somatic embryogenesis in rice (Oryza sativ L.)

Rice (Oryza sativa) embryogenic calli were obtained from mature zygotic embryos culture using two varieties of rice, Sakha 101 and Sakha104 on Murashige and Skoog (1962 ) medium supplemented with 1mg/l, 2mg/l and 3mg/l 2,4- dichlorophenoxy acetic acid. The results indicated that the best medium was that containing 3mg/l. Histological analysis of somatic embryogenesis revealed that after two weeks of culture of explants on the callus induction medium, somatic embryo development began with a cluster of proembryogenic cells in the peripheral region of the calli. The outer cell layer of embryogenic calli consisted of small and isodiametric cells, each with a dense cytoplasm and a prominent nucleus and nucleolus, whereas the inner cell layer is composed of large cells with small nuclei and large vacuoles. These embryogenic cells underwent a series of organized divisions and formed the proembryo with a well-defined protodermis. At 20 days after initiation of culture, these proembryos continued a series of organized divisions and gave rise to globular somatic embryos which are delimited by a well-defined layer of epidermal cells with conspicuous nuclei. These somatic embryos had no apparent vascular connection with the mother tissue and had a suspensor. After ten days of culture on regeneration medium, globular somatic embryos developed into heart-shaped somatic embryos, which consisted of cells with prominent nuclei and dense cytoplasm. After 15 days of culture on regeneration medium torpedo-shaped somatic embryos were observed. Then, after 25 to30 days, it turned into a monocotyledon form.

Titanium dioxide nanoparticles affect somatic embryo initiation, development, and biochemical composition in Paulownia sp. seedlings

Paulownia is a fast-growing tree with multiple uses. The present study was conducted to determine the most appropriate concentration of TiO2 nanoparticles (titanium dioxide) for the induction of somatic embryogenesis and regeneration, reduction of free radicals, and the increase of antioxidant enzymes. Variables examined included explants (leaf and petiole), Paulownia species (Paulownia shantung, Paulownia elongate and Paulownia fortune), and concentrations of TiO2 nanoparticle. The results showed significant effect of TiO2 on callus induction for all the three species. For Paulownia shantung, the callus formation for the samples in the medium containing 60 mg/L of TiO2 was 100% while for the samples in the control condition (without TiO2) it was 25%. The highest of somatic embryogenesis was observed for Paulownia shantung. However, the highest levels of flavonoids, anthocyanins, carotenoids, and chlorophyll a, b, and total chlorophyll were observed in induced and regenerated embryos at a concentration of 60 mg/L titanium dioxide nanoparticles. By increasing the concentration of TiO2, the amount of leaf soluble protein decreased. At the concentration of 60 mg/L TiO2 the density, number and regeneration of somatic embryo induction increased compared to the studied treatments. The largest ratio of seedling rooting was observed in ½MS (Reduction to half the entire MS medium content) supplemented with 1.5 mg/L IBA. The results can introduce lead to a novel protocol to proliferate healthy plantlets of Paulownia in a short time. Therefore, the present study led to the development of an effective and useful protocol for somatic embryo induction, regeneration, and increase of antioxidants in regenerated seedlings in the shortest time applying titanium dioxide concentration in the culture medium.

Global Transcriptome and Coexpression Network Analyses Reveal New Insights Into Somatic Embryogenesis in Hybrid Sweetgum (Liquidambar styraciflua × Liquidambar formosana).

Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Despite recent scientific headway in deciphering the difficulties of somatic embryogenesis, the overall picture of key genes, pathways, and co-expression networks regulating SE is still fragmented. Therefore, deciphering the molecular basis of somatic embryogenesis of hybrid sweetgum remains pertinent. In the present study, we analyzed the transcriptome profiles and gene expression regulation changes via RNA sequencing from three distinct developmental stages of hybrid sweetgum: non-embryogenic callus (NEC), embryogenic callus (EC), and redifferentiation. Comparative transcriptome analysis showed that 19,957 genes were differentially expressed in ten pairwise comparisons of SE. Among these, plant hormone signaling-related genes, especially the auxin and cytokinin signaling components, were significantly enriched in NEC and EC early. The K-means method was used to identify multiple transcription factors, including HB-WOX, B3-ARF, AP2/ERF, and GRFs (growth regulating factors). These transcription factors showed distinct stage- or tissue-specific expression patterns mirroring each of the 12 superclusters to which they belonged. For example, the WOX transcription factor family was expressed only at NEC and EC stages, ARF transcription factor was expressed in EC early, and GRFs was expressed in late SE. It was noteworthy that the AP2/ERF transcription factor family was expressed during the whole SE process, but almost not in roots, stems and leaves. A weighted gene co-expression network analysis (WGCNA) was used in conjunction with the gene expression profiles to recognize the genes and modules that may associate with specific tissues and stages. We constructed co-expression networks and revealed 22 gene modules. Four of these modules with properties relating to embryonic potential, early somatic embryogenesis, and somatic embryo development, as well as some hub genes, were identified for further functional studied. Through a combination analysis of WGCNA and K-means, SE-related genes including AUX22, ABI3, ARF3, ARF5, AIL1, AIL5, AGL15, WOX11, WOX9, IAA29, BBM1, MYB36, LEA6, SMR4 and others were obtained, indicating that these genes play an important role in the processes underlying the progression from EC to somatic embryos (SEs) morphogenesis. The transcriptome information provided here will form the foundation for future research on genetic transformation and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; Key genes obtained from coexpression network analysis at each critical stage of somatic embryo can be considered as potential candidate genes to verify these networks.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Exogenous Expression of pou5f3 Facilitates the Development of Somatic Cell Nuclear Transfer Embryos in Zebrafish at the Early Stage.

Enucleated oocytes can reprogram differentiated nuclei to totipotency after somatic cell nuclear transfer (SCNT), which is valuable in understanding nuclear reprogramming and generating genetically modified animals. To date, reprogramming efficiency is low and the development of SCNT embryos is not going as well as anticipated. To further disclose the reprogramming mechanisms during SCNT zebrafish embryo development, we examined the expression patterns of transcription regulation factors and regulated them by mRNA and morpholino microinjection. In this study, we show that stem cell-related transcription factors are downregulated in zebrafish SCNT embryos at the blastula stage. Exogenous expression of pou5f3 at the single-cell stage improves SCNT embryo development from the blastula to the gastrula stage. We also found that exogenous expression of klf4 or sox2 decreases SCNT embryo development from the blastula to the gastrula stage, while expression of nanog is necessary for the development of SCNT embryos. Our results conclude that zebrafish pou5f3 facilitates the development of SCNT embryos from the blastula to gastrula stage.

Similarities and Differences in the GFP Movement in the Zygotic and Somatic Embryos of Arabidopsis.

Intercellular signaling during embryo patterning is not well understood and the role of symplasmic communication has been poorly considered. The correlation between the symplasmic domains and the development of the embryo organs/tissues during zygotic embryogenesis has only been described for a few examples, including Arabidopsis. How this process occurs during the development of somatic embryos (SEs) is still unknown. The aim of these studies was to answer the question: do SEs have a restriction in symplasmic transport depending on the developmental stage that is similar to their zygotic counterparts? The studies included an analysis of the GFP distribution pattern as expressed under diverse promoters in zygotic embryos (ZEs) and SEs. The results of the GFP distribution in the ZEs and SEs showed that 1/the symplasmic domains between the embryo organs and tissues in the SEs was similar to those in the ZEs and 2/the restriction in symplasmic transport in the SEs was correlated with the developmental stage and was similar to the one in their zygotic counterparts, however, with the spatio-temporal differences and different PDs SEL value between these two types of embryos.

In silico characterization of putative gene homologues involved in somatic embryogenesis suggests that some conifer species may lack LEC2, one of the key regulators of initiation of the process

BackgroundSomatic embryogenesis (SE) is the process in which somatic embryos develop from somatic tissue in vitro on medium in most cases supplemented with growth regulators. Knowledge of genes involved in regulation of initiation and of development of somatic embryos is crucial for application of SE as an efficient tool to enable genetic improvement across genotypes by clonal propagation.ResultsCurrent work presents in silico identification of putative homologues of central regulators of SE initiation and development in conifers focusing mainly on key transcription factors (TFs) e.g. BBM, LEC1, LEC1-LIKE, LEC2 and FUSCA3, based on sequence similarity using BLASTP. Protein sequences of well-characterised candidates genes from Arabidopsis thaliana were used to query the databases (Gymno PLAZA, Congenie, GenBank) including whole-genome sequence data from two representative species from the genus Picea (Picea abies) and Pinus (Pinus taeda), for finding putative conifer homologues, using BLASTP. Identification of corresponding conifer proteins was further confirmed by domain search (Conserved Domain Database), alignment (MUSCLE) with respective sequences of Arabidopsis thaliana proteins and phylogenetic analysis (Phylogeny.fr).ConclusionsThis in silico analysis suggests absence of LEC2 in Picea abies and Pinus taeda, the conifer species whose genomes have been sequenced. Based on available sequence data to date, LEC2 was also not detected in the other conifer species included in the study. LEC2 is one of the key TFs associated with initiation and regulation of the process of SE in angiosperms. Potential alternative mechanisms that might be functional in conifers to compensate the lack of LEC2 are discussed.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives—such as trichostatin A—characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1–7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2–7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8–10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Inhibition of Suv39h1/2 expression improves the early development of Debao porcine somatic cell nuclear transfer embryos

Suppressor of variegation 3-9 homolog (Suv39h)1 and 2, Histone H3 lysine 9 trimethylation (H3K9me3)-specific methyltransferases, are mainly involved in regulating the dynamic changes of H3K9me3. Regulating Suv39h expression influences the early development of mice somatic cell nuclear transfer (SCNT) embryos, there are few reports concerning their features in domestic animals. The aim of the present study was to characterize the Suv39h function in early development of Debao porcine SCNT embryos. The global level of H3K9me3 and the expression profiles of Suv39h1/2 in porcine early embryos were analysed by immunohistochemistry and qRT-PCR methods, respectively. Their roles in cell proliferation and histone modification of Debao porcine foetal fibroblast cells (PFFs), and developmental competence of porcine SCNT embryos were investigated by shRNA technology. The methylation levels of H3K9me3 and the expression patterns of Suv39h1 and Suv39h2 were similar (p<.05), and both of them displayed higher levels in Debao porcine SCNT embryos compared with that in PA embryos. The global levels of H3K9me3 and the expressions of G9a, HDAC1 and DNMT1 were decreased by combined inhibition of Suv39h1 and Suv39h2 (p<.05), while the expression of HAT1 was increased (p<.05). Downregulation of Suv39h1/2 also promoted cell proliferation and resulted in a significant increase in the expression of CyclinA2, CyclinB and PCNA in PFFs (p<.05). Furthermore, the use of donor somatic nuclei which depleted H3K9me3 by inhibiting Suv39h1/2 expression markedly increased the cleavage rate, the blastocyst rate and the total cell number of blastocysts of Debao porcine SCNT embryos (p<.05). Altogether, the above results indicate that H3K9me3 levels and Suv39h1/2 expressions display similar patterns in porcine early embryo, and low levels of them are critical to cell proliferation of PFFs and early development of SCNT embryos.

Development of soybean somatic embryos under different light spectra

Soybean (Glycine max (L.) Merr. cv. 'Primorskaya 28') plantlet development from somatic embryos (embryoids) under different spectra was studied. Light irradiation with intensity of 48 μmol/(m2·s) was generated by LEDs (light emitting diodes) with various spectra: cold white (CW), white (W), warm white (WW), full spectrum (FS, 450 nm and 660 nm), red (R, 630 nm), blue (B, 440 nm), and sun box (SB, close to the sun spectrum at wavelength range 440–660 nm). Fluorescent lamps were used as a control illumination (K). Our results showed that experimental LED lights with different spectra possessed the potential to positively affect the development of soybean plantlets from embryoid phase to a full-fledged plantlet, ready to be transferred into soil. The best effect was observed for the FS, which provided development of the highest plantlets. SB light was promising after one month of cultivation, but provided no significant differences in plantlets growth at the end of the experiment. A tendency to the lower growth was observed for plantlets under CW, W, WW, and K. Red light did not provide notable shoot elongation, as was supposed to. Investigations will be continued to clarify and refine the obtained data on the effect of light spectra on soybean plantlet development from embryoids.

Developing in vitro selection methods to high temperature stress in pruacan and cacao

Increasing in the earth’s surface temperature due to global warming threatens the survival of plants in the sub-tropics and in the tropics areas. Looking for plant varieties that can adapt to the threat of global warming impacts is very important at this time. The availability of effective and efficient selection methods are needed to get it. This study aimed to develop an in vitro selection method for resistance to high temperature stress in two tropical plants: Pruacan (Pimpinella pruatjan Molk.) and Cacao (Theobroma cacao L.). The in vitro selection methods were developed by testing the embryogenic culture of pruacan and cacao somatic embryos development under various temperature incubations. The two plants showed a different level of tolerance to high-temperature stress. Temperatures of 35 °C for two months did not cause the death of cacao somatic embryos, but the temperature of 32 °C for 2 and 3 months had caused the death of pruacan somatic embryos by 80 and 90%, respectively. The 100% death of cacao somatic embryos occurred at 40 °C and 45 °C on days 16 and 5 of incubation, respectively. The in vitro selection for high-temperature stress in pruacan and cacao plants were at 32 °C and between 40-45 °C respectively. Developing in vitro selection methods in plant species is needed to face the challenges of climate change in the future.

In vitro somatic embryogenesis of Texas ebony (Ebenopsis ebano [Berland.] Barneby &amp; J.W. Grimes)

Texas ebony (Ebenopsis ebano [Berland.] Barneby & J.W. Grimes) is a member of the Fabaceae that is native to Mexico. Its wood has multiple uses, and its natural propagation is limited and only by seed. The present investigation describes the first report of Texas ebony somatic embryogenesis with the objective of identifying medium, growth regulator components, and culture conditions. Immature cotyledons were cultured on semisolid and in liquid media containing Murashige and Skoog (MS) basal salts plus 0.0 to 9.06 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The most frequent generation of embryogenic callus occurred with cotyledons cultured in the dark in liquid medium containing 9.06 μM 2,4-D. For the maturation phase, the embryogenic calluses were subcultured on semisolid MS medium with 3.78 to 30.24 μM abscisic acid (ABA), 3% and 6% polyethylene glycol (PEG) 4000, or 3 to 5% sucrose. Sucrose at 4% and 5% induced the greatest total number of somatic embryos; however, the maturation frequency and embryo quality were both higher with 6% PEG. Histological analysis revealed the ontogeny, morphology, and development of somatic embryos. Finally, the mature somatic embryos were subcultured for germination in half-strength MS medium plus 6-benzylaminopurine (BAP; 2.22 μM and 4.44 μM), gibberellic acid (GA3; 1.45 μM and 2.89 μM), or BAP (2.22 μM) and GA3 (1.45 μM). Embryo germination was superior with 2.22 μM BAP; however, radicle generation was poor in all of the treatments. This study demonstrated that for each phase of somatic embryogenesis, specific combinations of culture medium components and incubation environment are required.

In vitro Regeneration of Clematis Plants in the Nikita Botanical Garden via Somatic Embryogenesis and Organogenesis.

The effects of growth regulators, namely, 6-benzylaminopurine (BAP) and thidiazuron (TDZ), on the morphogenic capacity of 13 cultivars of clematis plants, in terms of their morphological structure formation, shoot regeneration, and somatic embryo development, are presented. The clematis cultivars ‘Alpinist,’ ‘Ay-Nor,’ ‘Bal Tsvetov,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Kosmicheskaya Melodiya,’ ‘Lesnaya Opera,’ ‘Madame Julia Correvon,’ ‘Nevesta,’ ‘Nikitsky Rosovyi,’ ‘Nikolay Rubtsov,’ ‘Serenada Kryma,’ and ‘Vechniy Zov’ were taken in collection plots of the Nikita Botanical Gardens for use in study. After explant sterilization with 70% ethanol (1 min), 0.3–0.4% Cl2 (15 min), and 1% thimerosal (10 min), 1-cm long segments with a single node were introduced to an in vitro culture. The explants were established on the basal MS medium supplemented with BAP (2.20–8.90 μM) and 0.049 μM NAA, or TDZ (3.0; 6.0, and 9.0 μM) with 30 g/L sucrose and 9 g/L agar. The medium with 0.89 μM BAP served as the control. Culture vessels and test tubes with the explants were maintained in plant growth chamber-controlled conditions: with a 16-h photoperiod, under cool-white light fluorescent lamps with a light intensity of 37.5 μmol m–2 s–1, at a temperature of 24 ± 1°C. Histological analysis demonstrated that adventitious bud and somatic embryo formation in studied clematis cultivars occurred at numerous areas of active meristematic cell zones. The main role of plant growth regulators and its concentrations were demonstrated. It was determined that maximum adventitious microshoot regeneration without any morphological abnormalities formed on the media supplemented with BAP or TDZ. 4.40 μM BAP, or 6.0 μM TDZ were optimal cytokinin concentrations for micropropagation. The explants of ‘Alpinist,’ ‘Ay-Nor,’ ‘Crimson Star,’ ‘Crystal Fountain,’ ‘Nevesta,’ and ‘Serenada Kryma’ cultivars displayed high morphogenetic capacity under in vitro culturing. During indirect somatic embryogenesis, light intensity 37.5 μmol m–2 s–1 stimulated a higher-number somatic embryo formation and a temperature of 26°C affected somatic embryo development. Active formation of primary and secondary somatic embryos was also demonstrated. 2.20 μM BAP with 0.09 μM IBA affected the high-number somatic embryo formation for eight cultivars. Secondary somatic embryogenesis by the same concentration of BAP was induced. The frequency of secondary somatic embryogenesis was higher in ‘Crystal Fountain’ (100%), ‘Crimson Star’ (100%), ‘Nevesta’ (97%), and ‘Ay-Nor’ (92%) cultivars. Based on these results, the methodology for direct somatic embryogenesis and organogenesis of studied clematis cultivars has been developed.

Mitochondrial bioenergetics and enzymatic antioxidant defense differ in Paraná pine cell lines with contrasting embryogenic potential

Araucaria angustifolia is classified as a critically endangered species by the International Union for Conservation of Nature. This threat is worsened by the inefficiency of methods for ex-situ conservation and propagation. In conifers, somatic embryogenesis (SE) associated with cryopreservation is an efficient method to achieve germplasm conservation and mass clonal propagation. However, the efficiency of SE is highly dependent on genotype responsivity to the artificial stimulus used in vitro during cell line proliferation and later during somatic embryo development. In this study, we evaluated the activity of antioxidant enzymes and characterized mitochondrial functions during the proliferation of embryogenic cells of A. angustifolia responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The activities of the antioxidant enzymes GR (EC 1.6.4.2), MDHAR (EC 1.6.5.4), and POX (EC 1.11.1.7) were increased in SE1 culture, while in SE6 culture, only the activity of DHAR (EC 1.8.5.1) was significantly higher. Additionally, SE6 culture presented a higher number of mitochondria, which agreed with the increased rate of oxygen consumption compared to responsive SE1 culture; however, the mitochondrial volume was lower. Although the ATP levels did not differ, the NAD(P)H levels were higher in SE1 cells. NDs, AOX, and UCP were less active in responsive SE1 than in non-responsive cells. Our results show significant differences between SE1 and SE6 embryogenic cells regarding mitochondrial functions and antioxidant enzyme activities, which may be intrinsic to the in vitro proliferation phase of both cell lines, possessing a crucial role for the induction of in vitro maturation process.

Development of Porcine Somatic Cell Nuclear Transfer Embryos Following Treatment Time of Endoplasmic Reticulum Stress Inhibitor.

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 μM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.