Somatic Embryogenesis Process Research Articles

Machine Learning based Sorting of Somatic Embryos for In-Line processing in Automated SE Fluidics System

Somatic embryogenesis (SE) can be a viable method for the clonal propagation of many economically significant forest trees, particularly coniferous trees like pines and spruces. However, large-scale production of SE plants requires automation to reduce manual labor and attain cost-efficiency. The most labor-intensive step of the SE process for SE plant production is selecting and harvesting mature embryos. Embryo maturation is not a synchronized process; selecting the most developed embryos capable of continuous development is necessary. However, there needs to be more research conducted on mapping morphological features to germination-competent mature somatic embryos. This paper lays down the preliminary work of employing machine learning techniques for classifying large volumes of images of mature somatic embryos processed using an automated SE processing system based on fluidics processing referred to as SE Fluidics system. The results show that machine learning could be an alternative classification methodology instead of the traditional manual morphology-based classification process based on image analysis. The paper discusses two popular image classification techniques, namely Convolution Neural Network (CNN) and Support Vector Machine (SVM), applying them to both binary (black and white) and grayscale images. It is observed that grayscale images provide better accuracy with the SVM technique and outperform morphology-based classification in terms of processing speed (17.6% faster) across the test envelope. On the other hand, CNN-based classification shows better processing speeds only at a lower number of convolution layers. Hence, the data scientist can optimally select the number of convolution layers to get the desired accuracy-processing speed combination.

Influence of exogenous polyamines on the secondary somatic embryogenesis of cork oak (Quercus suber L.)

ABSTRACT Quercus suber L. is the main woody tree species in the Mediterranean basin. The in vitro regeneration from adult material, through primary somatic embryogenesis, is a well-known process, but the use of secondary somatic embryos for plant regeneration remains a very sparsely studied process. The main objective of this work is to explore the cork oak regeneration potential by using the secondary somatic embryogenesis process. Mainly, in this work, we report the polyamine effect. Explants used consisted on primary mature embryos, derived from leaves rejuvenated by epicormic shoot of the Moroccan Quercus suber. Three different polyamines were added to the basal medium, which was composed by macronutrients of N 30 K, 30 g/l glucose, and 7 g/l agar. Three polyamines, Putrescine, Spermine, and Spermidine, were added to the basal medium at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/l. Explants were tested after 8 weeks. Morphological analysis showed that the medium with 0.4 mg/l Spermidine provided the best result for secondary embryos, which corresponds to a very significant (p < 0.05) increase of 375%. The number of secondary embryos directly formed was 2.70 ± 0.51. Similarly, the optimum concentrations for high number of clusters (0.50 ± 0.11) and embryo clusters (1.43 ± 0.35) were increased by 145% and 158%. The addition of the polyamine also acted on the quality of embryos formed. A very significant (p < 0.05) increase in the size of secondary embryos was observed compared to the medium without polyamines. Spermidine showed the greatest increase (about 38%).

EVALUACIÓN DEL EFECTO DE LA PUTRESCINA EXÓGENA DURANTE LA MADURACIÓN Y GERMINACIÓN in vitro DE EMBRIONES SOMÁTICOS EN DOS GENOTIPOS DE AGAVE

&lt;p&gt;&lt;strong&gt;Background.&lt;/strong&gt; Agaves are an important economic source in industrial products, in the food and pharmaceutical industry, steroidal animal feed, ornamental plants and as a material for biofuels. Despite their economic importance, some species present problems in their conventional propagation, which makes their reproduction difficult and has caused some species to be in some risk category. Therefore, there is a need to develop efficient protocols for &lt;em&gt;in vitro&lt;/em&gt; micropropagation via somatic embryogenesis. The positive effect of the application of exogenous putrescine on the maturation and germination of somatic embryos is known. &lt;strong&gt;Objective&lt;/strong&gt;. To determine the effect of exogenous putrescine on the maturation and germination of somatic embryos of &lt;em&gt;A. cupreata&lt;/em&gt; and &lt;em&gt;A. angustifolia&lt;/em&gt;.&lt;strong&gt; Methodology&lt;/strong&gt;. Two concentrations of putrescine (100 and 150 mg/L) and two controls, one positive and one negative, were evaluated in the somatic embryo maturation medium and later in the germination of somatic embryos. &lt;strong&gt;Results&lt;/strong&gt;. Putrescine favors the maturation and germination of somatic embryos in both species. &lt;strong&gt;Implications&lt;/strong&gt;. The results of this research contribute to a better understanding of the process of somatic embryogenesis and open the possibility of applying putrescine in the process of somatic embryogenesis at a commercial level. &lt;strong&gt;Conclusion&lt;/strong&gt;. Exogenous putrescine in culture medium significantly improves the maturation and germination of somatic embryos in &lt;em&gt;Agave&lt;/em&gt;.&lt;/p&gt;

Changes and transcriptome regulation of endogenous hormones during somatic embryogenesis in Ormosia henryi Prain.

Ormosia henryi is a rare and endangered plant growing in southern China. Somatic embryo culture is an effective measure for the rapid propagation of O. henryi. It has not been reported how regulatory genes induce somatic embryogenesis by regulating endogenous hormone changes during the process of somatic embryogenesis in O. henryi. In this study, we analysed the endogenous hormone levels and transcriptome data of nonembryogenic callus (NEC), embryogenic callus (EC), globular embryo (GE) and cotyledon embryo (CE) in O. henryi. The results showed that the indole-3-acetic acid (IAA) content was higher and the cytokinins (CKs) content was lower in EC than in NEC, and the gibberellins (GAs) and abscisic acid (ABA) contents were significantly higher in NEC than in EC. The contents of IAA, CKs, GAs and ABA increased significantly with EC development. The expression patterns of differentially expressed genes (DEGs) involved in the biosynthesis and signal transduction of auxin (AUX) (YUCCA and SAUR), CKs (B-ARR), GAs (GA3ox, GA20ox, GID1 and DELLA) and ABA (ZEP, ABA2, AAO3, CYP97A3, PYL and ABF) were consistent with the levels of endogenous hormones during somatic embryogenesis (SE). In this study, 316 different transcription factors (TFs) regulating phytohormones were detected during SE. AUX/IAA were downregulated in the process of EC formation and GE differentiation into CE, but other TFs were upregulated and downregulated. Therefore, we believe that relatively high IAA content and low CKs, GAs and ABA contents contribute to EC formation. The differential expression of AUX, CKs, GAs and ABA biosynthesis and signal transduction genes affected the endogenous hormone levels at different stages of SE in O. henryi. The downregulated expression of AUX/IAA inhibited NEC induction, promoted EC formation and GE differentiation into CE.

Response of Morphogenesis and Cell Proliferation to Allelopatic Compounds on Rice Germplasm

Allelopathic compounds are chemical substances produced by weeds, including Imperata cylindrica, that can inhibit the absorption of nutrients and cell growth of neighboring plants. Tissue culture technique can be used to determine the effect of media composition on morphogenesis and cell proliferation, as indicated by related genes. Through the process of somatic embryogenesis, cells regenerate into whole plants via several phases initiated by gene expression. Genes are the signal that connects environmental cues and plant cells. The objective of this study was to determine the effect of allelopathic compounds from Imperata cylindrica root extract on the morphogenesis and cell proliferation of indigenous Indonesian rice plant. The used callus was derived from the seeds of Bondoyudo, Caok, Ciliwung and Situbagendit rice varieties. The results of callus induction were selected and transferred to regeneration media that had been treated with I. cylindrica roots extract of 2.5 g/L and 5 g/L. The development of callus was monitored weekly for six weeks. As for the molecular analysis, four-week-old callus was utilized. The results demonstrated that the root extract of Imperata cylindrica had a specific effect on the morphogenesis and proliferation of rice cells depending on concentration and target plant. At a molecular level, the expression of the OsBBM, OsLEA, OsLEC1, OsSERK, and OsWOX4 genes was affected differently by the administration of allelopathic compounds at a concentration of 5 g/L on a molecular level.

Secondary somatic embryogenesis in Cork oak: influence of plant growth regulators

Cork oak (Quercus suber L.) is one of the most important Mediterranean forest tree species, and the Maâmora forest-Morocco is considered to be the world largest cork oak stand. Clonal propagation of Quercus suber via somatic embryogenesis is an alternative to conventional tree propagation methods. However, complete maturation of somatic embryos is considered the major bottleneck for mass propagation of Quercus suber during somatic embryogenesis. This study focuses on influences of cytokinins on secondary somatic embryogenesis. Cytokinins showed a negative effect on the induction of secondary somatic embryogenesis. For the various concentrations tested, the number of secondary somatic embryos was significantly reduced except in the case with low dose of 2iP, which showed a slight increase. Abscisic acid (ABA) may be beneficial at low concentrations between 0.3 and 0.5 mg/l. The best number of secondary embryos was given at 0.5 mg/l ABA with an average number of 5 embryos from each primary embryo, which represents a slight increase below 10% compared to control medium. Also, we found that the number of responsive embryos presenting secondary somatic embryos based on their cotyledons decreased once adding ABA. Phenylurea derivatives TDZ and DPU do not promoted the process of secondary somatic embryogenesis. DPU showed no significant effect with a similar response to that of the control medium and TDZ showed an inhibitory effect. Gibberellic acid (GA3) partially blocked the process of somatic embryos multiplication, but promoted remarkably the germination of cotyledonary embryos without requesting a particular protocol. Our results provide a new insight into embryo development, establishing the basis for further research toward improvement of secondary somatic embryogenesis in Moroccan cork oak.

Development of a testing system for regeneration regulators in &lt;i&gt;Pisum sativum&lt;/i&gt; L.

Pisum sativum L. (pea) is one of the most important agricultural crops, because its seeds have high protein content, and, due to its ability have symbiotic relationships with nitrogen-fixing bacteria, these plants need less fertilizers. Nevertheless, we are faced with the need to improve old and create new methods for obtaining novel varieties of peas and other agricultural plants. The formation of regenerated pea plants is difficult to achieve in the in vitro culture. Accordingly, transformation of this species is a laborious process. In this regard, the search for morphogenic regulators of somatic embryogenesis (SE) in pea is an urgent problem. A number of publications reported on the genes regulating the SE process in a model plant from the legume family, Medicago truncatula [1]. In our study, we search for the in vitro cultivation system in peas, suitable to test the effect of putative SE regulators in this species. We tested several pea transformation techniques using different explant variants: embryonic axes from mature and immature seeds, as well as shoot apexes. Out of the tested options, the transformation of mature seeds turned out to be optimal. We also designed a set of DNA constructs in silico, which are suitable for the search of morphogenic regulators in peas.

The source, level, and balance of nitrogen during the somatic embryogenesis process drive cellular differentiation.

Since the discovery of somatic embryogenesis (SE), it has been evident that nitrogen (N) metabolism is essential during morphogenesis and cell differentiation. Usually, N is supplied to cultures in vitro in three forms, ammonium (NH4+), nitrate (NO3-), and amino N from amino acids (AAs). Although most plants prefer NO3- to NH4+, NH4+ is the primary form route to be assimilated. The balance of NO3- and NH4+ determines if the morphological differentiation process will produce embryos. That the N reduction of NO3- is needed for both embryo initiation and maturation is well-established in several models, such as carrot, tobacco, and rose. It is clear that N is indispensable for SE, but the mechanism that triggers the signal for embryo formation remains unknown. Here, we discuss recent studies that suggest an optimal endogenous concentration of auxin and cytokinin is closely related to N supply to plant tissue. From a molecular and biochemical perspective, we explain N's role in embryo formation, hypothesizing possible mechanisms that allow cellular differentiation by changing the nitrogen source.

Phenotypically abnormal cotyledonary Vitis vinifera embryos differ in anatomy, endogenous hormone levels and transcriptome profiles.

In many perennial fruit species, including grapevine (Vitis vinifera L.), the highly complex process of somatic embryogenesis (SE) can result in the formation of a deformed embryo, although the underlying reasons are still poorly understood. Here, V. vinifera cv. 'Chardonnay' cotyledonary embryos with distinct morphologies were used to address this issue. Normal cotyledonary embryos (NCEs) and elongated cotyledonary embryos (ECEs) were observed to have better-developed vasculature and shoot meristems than the vitrified cotyledonary embryos (VCEs) and fused cotyledonary embryos (FCEs), but ECEs were less developed. We determined that the morphological differences in these phenotypically abnormal embryos were likely associated with endogenous hormone levels, since concentrations of the phytohormones indoleacetic acid (IAA) and abscisic acid (ABA) in NCEs were higher than in the other three types. Comparative transcriptome analysis revealed large differences in gene expression of the hormone signaling pathways in normal and abnormal cotyledonary embryos. Weighted gene co-expression network analysis of the different cotyledonary types allowed the identification of co-regulated gene modules associated with SE, suggesting a role for ERF family genes and other transcription factors (TFs) in regulating morphology. Moreover, an analysis of morphology-specific gene expression indicated that the activation of a specific protein kinase, small heat shock proteins (sHSPs) and certain TFs was closely associated with the formation of normal cotyledonary embryos. Our comparative analyses provide insights into the gene networks regulating somatic cotyledon development and open new avenues for research into plant regeneration and functional genomic studies of malformed embryos.

Gibberellic acid influences growth indices and biochemical parameters in micropropagated Ocimum gratissimum L. explants

Background: Ocimum gratissimum L. is a well-known tropical and subtropical plant widely utilised for both medicinal and nutritional purposes. However, its continuous existence is currently threatened because of excessive human exploitation and non-regulated collection.Aim: This study aimed at mass-producing O. gratissimum through its explants cultured on Murashige and Skoog (MS) medium.Settings: Sterile explants of O. gratissimum were used to generate whole plant through the process of somatic embryogenesis and under the influence of different plant growth regulators (PGRs).Methods: The growth medium was supplemented with various concentrations (0.1 mg/L, 0.5 mg/L and 1.0 mg/L) of PGRs, namely, gibberellic acid (GA3) and benzyl aminopurine (BAP) in combination with 0.2 mg/L indole acetic acid (IAA), which also served as the control.Results: The results showed that germination response, shoot and root lengths were significantly enhanced in O. gratissimum explants raised in media containing the two PGRs in a concentration-dependent manner after four weeks of culture. Ocimum gratissimum explants treated with GA3 and BAP also experienced reduced lipid peroxidation and ascorbic acid concentration, particularly at the highest tested concentration (1.0 mg/L) as evidenced by the significant drop in malondialdehyde (MDA) concentration. In response to this, the activities of superoxide dismutase (SOD) and catalase (CAT) were significantly increased in O. gratissimum explants raised in MS medium supplemented with PGRs.Conclusion: These results generally suggest that GA3 in combination with IAA is more favourable than BAP for the micropropagation of O. gratissimum explants. Thus, our study revealed that PGRs possess special attributes, which could be exploited in tissue culture for the micropropagation of O. gratissimum explants.

Thermopriming-associated proteome and sugar content responses in Pinus radiata embryogenic tissue

Improving the capacity of plants to face adverse environmental conditions requires a deep understanding of the molecular mechanisms governing stress response and adaptation. Proteomics, combined with metabolic analyses, offers a wide resource of information to be used in plant breeding programs. Previous studies have shown that somatic embryogenesis in Pinus spp. is a suitable tool not only to investigate stress response processes but also to modulate the behaviour of somatic plants. Based on this, the objective of this study was to analyse the protein and soluble sugar profiles of Pinus radiata embryonal masses after the application of high temperatures to unravel the mechanisms involved in thermopriming and memory acquisition at early stages of the somatic embryogenesis process. Results confirmed that heat provokes deep readjustments in the life cycle of proteins, together with a significant reduction in the carbon-flux of central-metabolism pathways. Heat-priming also promotes the accumulation of proteins involved in oxidative stress defence, in the synthesis of specific amino acids such as isoleucine, influences cell division, the organization of the cytoskeleton and cell-walls, and modifies the levels of free soluble sugars like glucose or fructose. All this seems to be regulated by proteins linked with epigenetic, transcriptional and post-transcriptional mechanisms.

Establishing Tetraploid Embryogenic Cell Lines of Magnolia officinalis to Facilitate Tetraploid Plantlet Production and Phenotyping.

The production of synthetic polyploids for plant breeding is compromised by high levels of mixoploids and low numbers of solid polyploid regenerants during in vitro induction. Somatic embryogenesis could potentially contribute to the maximization of solid polyploid production due to the single cell origin of regenerants. In the present study, a novel procedure for establishing homogeneous tetraploid embryogenic cell lines in Magnolia officinalis has been established. Embryogenic cell aggregate (ECA) about 100–200 μm across, and consisting of dozens of cells, regenerated into a single colony of new ECAs and somatic embryos following colchicine treatment. Histological analysis indicated that the few cells that survived some colchicine regimes still regenerated to form a colony. In some colonies, 100% tetraploid somatic embryos were obtained without mixoploid formation. New granular ECA from single colonies with 100% tetraploid somatic embryos were isolated and cultured individually to proliferate into cell lines. These cell lines were confirmed to be homogeneous tetraploid by flow cytometry. Many tetraploid somatic embryos and plantlets were differentiated from these cell lines and the stability of ploidy level through the somatic embryogenesis process was confirmed by flow cytometry and chromosome counting. The establishment of homogeneous polyploid cell lines, which were presumed to represent individual polyploidization events, might expand the phenotypic variations of the same duplicated genome and create novel breeding opportunities using newly generated polyploid plantlets.

Genome-wide Characterization of the JmjC Domain-Containing Histone Demethylase Gene Family Reveals GhJMJ24 and GhJMJ49 Involving in Somatic Embryogenesis Process in Cotton.

The Jumonji C (JmjC) domain-containing protein family, an important family of histone demethylase in plants, can directly reverse histone methylation and play important roles in various growth and development processes. In the present study, 51 JmjC genes (GhJMJs) were identified by genome-wide analysis in upland cotton (Gossypium hirsutum), which can be categorized into six distinct groups by phylogenetic analysis. Extensive syntenic relationship events were found between G. hirsutum and Theobroma cacao. We have further explored the putative molecular regulatory mechanisms of the JmjC gene family in cotton. GhJMJ24 and GhJMJ49 were both preferentially expressed in embryogenic callus compared to nonembryogenic callus in cotton tissue culture, which might be regulated by transcription factors and microRNAs to some extent. Further experiments indicated that GhJMJ24 and GhJMJ49 might interact with SUVH4, SUVH6, DDM1, CMT3, and CMT1 in the nucleus, potentially in association with demethylation of H3K9me2. Taken together, our results provide a foundation for future research on the biological functions of GhJMJ genes in cotton, especially in somatic embryogenesis in cotton tissue culture, which is crucial for the regeneration of transgenic plants.

The SAUR gene family in coffee: genome-wide identification and gene expression analysis during somatic embryogenesis.

Small auxin-up RNA (SAUR) genes form a wide family supposedly involved in different physiological and developmental processes in plants such as leaf senescence, auxin signaling and transport, hypocotyl development and tolerance to abiotic stresses. The transcription of SAUR genes is quickly induced by auxins, a group of phytohormones of major importance on embryo development. To better understand the distribution and expression profile of such still not explored family in Coffea sp., especially during the development of somatic embryogenesis (SE), SAUR members were characterized in silico using the available Coffea canephora genome data and analyzed for gene expression by RT-qPCR in C. arabica embryogenic samples. Over C. canephora genome 31 CcSAURs were distributed by 11 chromosomes. Out of these 31 gene members, 5 SAURs were selected for gene expression analysis in C. arabica embryogenic materials. CaSAUR12 and CaSAUR18 were the members highly expressed through almost all plant materials. The other genes had more expression in at least one of the developing embryo stages or plantlets. The CaSAUR12 was the only member to exhibit an increased expression in both non-embryogenic calli and the developing embryo stages. The identification of SAUR family on C. canephora genome followed by the analysis of gene expression profile across coffee somatic embryogenesis process on C. arabica represents a further additional step towards a better comprehension of molecular components acting on SE. Along with new research about this gene family such knowledge may support studies about clonal propagation methods via somatic embryogenesis to help the scientific community towards improvements into coffee crop.

Identification and expression analysis of histone modification gene (HM) family during somatic embryogenesis of oil palm

BackgroundOil palm (Elaeis guineensis, Jacq.) is an important vegetable oil-yielding plant. Somatic embryogenesis is a promising method to produce large-scale elite clones to meet the demand for palm oil. The epigenetic mechanisms such as histone modifications have emerged as critical factors during somatic embryogenesis. These histone modifications are associated with the regulation of various genes controlling somatic embryogenesis. To date, none of the information is available on the histone modification gene (HM) family in oil palm.ResultsWe reported the identification of 109 HM gene family members including 48 HMTs, 27 HDMs, 13 HATs, and 21 HDACs in the oil palm genome. Gene structural and motif analysis of EgHMs showed varied exon–intron organization and with conserved motifs among them. The identified 109 EgHMs were distributed unevenly across 16 chromosomes and displayed tandem duplication in oil palm genome. Furthermore, relative expression analysis showed the differential expressional pattern of 99 candidate EgHM genes at different stages (non-embryogenic, embryogenic, somatic embryo) of somatic embryogenesis process in oil palm, suggesting the EgHMs play vital roles in somatic embryogenesis. Our study laid a foundation to understand the regulatory roles of several EgHM genes during somatic embryogenesis.ConclusionsA total of 109 histone modification gene family members were identified in the oil palm genome via genome-wide analysis. The present study provides insightful information regarding HM gene’s structure, their distribution, duplication in oil palm genome, and also their evolutionary relationship with other HM gene family members in Arabidopsis and rice. Finally, our study provided an essential role of oil palm HM genes during somatic embryogenesis process.

In Vitro Stress-Mediated Somatic Embryogenesis in Plants.

Somatic embryogenesis (SE) serves as a key biological model for studying cell totipotency and the ontogenic processes of zygotic embryogenesis in plants. The SE process, under in vitro conditions, can be induced from different sources of explant cultivated in a culture medium with plant growth regulators (PGR) or by subjecting tissues to abiotic stress treatments. Somatic embryogenesis, in plant tissue culture (PTC), is a multifactorial event. The use of PGR, particularly auxins, is an important factor during induction. However, in vitro abiotic stress treatments are physiologically, biochemically, and genetically relevant and should be further studied.

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.

Somatic Embryogenesis in Pines.

Among the different in vitro culture techniques, somatic embryogenesis has been one of the most important developments for plant tissue culture; it has enabled mass propagation and the development of biotechnological tools to enhance the productivity and quality of plantation forestry. This propagation technique together with cryopreservation is the base of multivarietal forestry.The development of somatic embryogenesis in forest trees dates from 1985, and in the last years several studies have focused on the development and optimization of the conifer somatic embryogenesis process to make it more efficient in terms of both the quantity and the characteristics of the plants obtained. However, these advances are not sufficiently refined to be implemented commercially for many Pinus spp. due to the high cost of the process derived from hand labor. Nowadays, trying to add value to the plants produced to compensate the high costs of the process, different studies are being developed in order to obtain Pinus somatic plants with better adaptation to environmental stresses prompted by the current situation of climate change.In this chapter, a summary of the recent somatic embryogenesis systems developed to achieve Pinus spp. high quality plants is presented.

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.

Heat Stress in Pinus halepensis Somatic Embryogenesis Induction: Effect in DNA Methylation and Differential Expression of Stress-Related Genes.

In the current context of climate change, plants need to develop different mechanisms of stress tolerance and adaptation to cope with changing environmental conditions. Temperature is one of the most important abiotic stresses that forest trees have to overcome. Recent research developed in our laboratory demonstrated that high temperatures during different stages of conifer somatic embryogenesis (SE) modify subsequent phases of the process and the behavior of the resulting ex vitro somatic plants. For this reason, Aleppo pine SE was induced under different heat stress treatments (40 °C for 4 h, 50 °C for 30 min, and 60 °C for 5 min) in order to analyze its effect on the global DNA methylation rates and the differential expression of four stress-related genes at different stages of the SE process. Results showed that a slight decrease of DNA methylation at proliferating embryonal masses (EMs) can correlate with the final efficiency of the process. Additionally, different expression patterns for stress-related genes were found in EMs and needles from the in vitro somatic plants obtained; the DEHYDRATION INDUCED PROTEIN 19 gene was up-regulated in response to heat at proliferating EMs, whereas HSP20 FAMILY PROTEIN and SUPEROXIDE DISMUTASE [Cu–Zn] were down-regulated in needles.