Somatic Embryos Research Articles

Lysophosphatidic acid improves development of porcine somatic cell nuclear transfer embryos

Lysophosphatidic acid improves development of porcine somatic cell nuclear transfer embryos

Protocol for plant regeneration from encapsulated somatic embryos of Citrus sinensis L.

BackgroundCitrus is an important fruit crop worldwide; artificial seeds are through encapsulation techniques of somatic embryos having many applications such as large-scale propagation and germplasm conservation. The aim of this study is the investigation of the viability of encapsulated citrus somatic embryos after different storage periods and to convert them into plantlets.ResultsCotyledonary-stage somatic embryos (5–7-mm size) regenerated from stigma explants of Washington navel orange (Citrus sinensis L.) were encapsulated individually in 3% sodium alginate. After different preservation periods (1, 2 or 3 months) at 10 °C, the encapsulated somatic embryos were cultured on Murashige and Skooge (MS) medium solidified with 7 g/l agar and supplemented with 50 g/l sucrose for germination. Percentage of regrowth, germination percentage, number of plantlets, plantlets height and number of leaves/shoot were recorded after different recovery periods. The germination percentages of encapsulated embryos were 90, 62.5 and 40% with storage for one, two and three months, respectively. Encapsulated somatic embryos preserved for 1 month developed the highest number of plantlets, while those preserved for 2 months developed the highest length of plantlets and the highest number of leaves/shoot after a recovery period of 10 months. Molecular analysis was performed of plantlets recovered from somatic embryos after preservation by encapsulation, and the results showed that the percentages of polymorphism were 7.7% with the two primers in all treatments.ConclusionsIt could be concluded that callus developed from stigma explants was able to regenerate indirect somatic embryogenesis after 3 months. Cotyledonary-stage somatic embryos of citrus were successfully preserved by encapsulation using 3% sodium alginate. Frequencies of germination of encapsulated somatic embryos increased with increasing the recovery period on the germination medium. A maximum recovery frequency of 60.8% was obtained from encapsulated somatic embryos cultured on germination medium for up to 10 months. Also, recovery frequency of 62.5% was noticed from encapsulated somatic embryos preserved for 2 months at 10 °C. In addition, results indicated that recovered somatic embryos obtained from encapsulated somatic embryos were able to convert to normal plantlets.

Characterisation of hybrid larch somatic embryo maturation by biochemical analyses and by a novel, fast mid-infrared method.

The morphology of somatic embryos (SE) is not a sufficient criterion to determine the level of maturation and the optimal stage to transfer embryos for germination, unlike the biochemical components. This composition characterization in the laboratory is too restrictive to be considered at each maturation cycle, as would be necessary. It is, therefore, essential to consider alternative methods. The objectives of this work were to achieve a complete biochemical characterization of the embryos during their development, to serve as a reference, and develop a characterization based on infrared spectrometry and chemometrics. During the precotyledonary stage (0-3 weeks of maturation), water content and glucose and fructose levels were high, which is consistent with SE development. After 4 weeks, the cotyledonary SE had a metabolism oriented towards the storage accumulation of lipids, proteins and starch, whereas raffinose only appeared from 8 weeks. Mid-infrared calibration models were developed for water, proteins, lipids, carbohydrates, glucose, fructose, inositols, raffinose, stachyose, and starch contents with an r2 average of 0.84. A model was also developed to discriminate the weeks of SE maturation. Different classes of age were discriminated with at least 72% of accuracy. Infrared analysis of the SE based on their full biochemical spectral fingerprint revealed a very slight variation in composition between 7 and 9 weeks, information that is very difficult to obtain by conventional analysis methods. These results provide novel insights into the maturation of conifer SE and indicate that mid-infrared spectrometry could be an easy and effective method for SE characterization. This article is protected by copyright. All rights reserved.

Analysis of polycomb repressive complex 2 (PRC2) subunits in Picea abies with a focus on embryo development

BackgroundConserved polycomb repressive complex 2 (PRC2) mediates H3K27me3 to direct transcriptional repression and has a key role in cell fate determination and cell differentiation in both animals and plants. PRC2 subunits have undergone independent multiplication and functional divergence in higher plants. However, relevant information is still absent in gymnosperms.ResultsTo launch gymnosperm PRC2 research, we identified and cloned the PRC2 core component genes in the conifer model species Picea abies, including one Esc/FIE homolog PaFIE, two p55/MSI homologs PaMSI1a and PaMSI1b, two E(z) homologs PaKMT6A2 and PaKMT6A4, a Su(z)12 homolog PaEMF2 and a PaEMF2-like fragment. Phylogenetic and protein domain analyses were conducted. The Esc/FIE homologs were highly conserved in the land plant, except the monocots. The other gymnospermous PRC2 subunits underwent independent evolution with angiospermous species to different extents. The relative transcript levels of these genes were measured in endosperm and zygotic and somatic embryos at different developmental stages. The obtained results proposed the involvement of PaMSI1b and PaKMT6A4 in embryogenesis and PaKMT6A2 and PaEMF2 in the transition from embryos to seedlings. The PaEMF2-like fragment was predominantly expressed in the endosperm but not in the embryo. In addition, immunohistochemistry assay showed that H3K27me3 deposits were generally enriched at meristem regions during seed development in P. abies.ConclusionsThis study reports the first characterization of the PRC2 core component genes in the coniferous species P. abies. Our work may enable a deeper understanding of the cell reprogramming process during seed and embryo development and may guide further research on embryonic potential and development in conifers.

Transcriptome Landscape Analyses of the Regulatory Network for Zygotic Embryo Development in Paeonia ostii.

Paeonia ostii is a worldwide ornamental flower and an emerging oil crop. Zyotic embryogenesis is a critical process during seed development, and it can provide a basis for improving the efficiency of somatic embryogenesis (SE). In this study, transcriptome sequencing of embryo development was performed to investigate gene expression profiling in P. ostii and identified Differentially expressed genes (DEGs) related to transcription factors, plant hormones, and antioxidant enzymes. The results indicated that IAA (Indole-3-acetic acid), GA (Gibberellin), BR (Brassinosteroid) and ETH (Ethylene) were beneficial to early embryonic morphogenesis, while CTK (Cytokinin) and ABA (Abscisic Acid) promoted embryo morphogenesis and maturation. The antioxidant enzymes' activity was the highest in early embryos and an important participant in embryo formation. The high expression of the genes encoding fatty acid desaturase was beneficial to fast oil accumulation. Representative DEGs were selected and validated using qRT-PCR. Protein-protein interaction network (PPI) was predicted, and six central node proteins, including AUX1, PIN1, ARF6, LAX3, ABCB19, PIF3, and PIF4, were screened. Our results provided new insights into the formation of embryo development and even somatic embryo development in tree peonies.

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.).

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

Using of Somatic Embryogenesis for Improving Bolting-resistant New Local Cultivars of Heirloom Carrot Cultivars (Daucus carota) Under Egyptian Conditions

Foreign carrot varieties with desirable morphological traits for the local and global markets must bloom in low temperatures for a long period of time in Egypt, but these climatic conditions are not available for flowering and seed production for these varieties, so it is common to import carrot seeds from abroad. On the other hand, Local cultivars bloom earlier when planted later than the advised date. Due to the heat, it cannot be grown throughout the summer. Pushing the somatic cells to divide and create a new polarity is necessary for the induction of tissues in the lab and the creation of somatic embryos. It was shown that several auxins play a crucial role in the development of these embryos, particularly 2, 4-D (2,4-dichlorophenoxyacetic acid). This experiment was carried out to investigate the effect of five procedures in a tissue culture laboratory using two concentrations of 2, 4-D at a concentration of 2.5 and 5 mg/l for indirect somatic embryos induction from sexual embryos produced from culturing carrot (Daucus carota) seeds of Carrot cv. Kuroda (an heirloom variety). The results confirmed by scanning electron microscopy showed that it is crucial to compare the various processes used to create somatic Embryogenesis to assess their effectiveness. The four stages of somatic embryos, globular, heart, torpedo, and cotyledon form, were observed on the formed callus. The highest amounts of callus fresh weight, the number of globular-shaped stage embryos, the number of heart-shaped stage embryos, the number of torpedo-shaped stage embryos, the number of cotyledon-shaped stage embryos, and the number of seedlings had been recorded in the media supplemented with 2, 4-D at a concentration of 5 mg. This study was able to produce somatic embryos of the foreign carrot cv. Kuroda, Plants obtained from somatic embryos are included in selection programs to produce varieties resistant to early flowering under Egyptian conditions. These results constitute a step in the way in the field of improving bolting-resistant new local cultivars of heirloom carrot cultivars (Daucus carota) under Egyptian conditions.

Establishment of an Efficient Somatic Embryogenesis Protocol for Giant Reed (Arundo donax L.) and Multiplication of Obtained Shoots via Semi-Solid or Liquid Culture

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h.

Blastocyst cell number and allocation affect the developmental potential and transcriptome of bovine Somatic Cell Nuclear Transfer embryos.

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. While the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117±6 total cells, whereas embryos with an average of 128±5 cells did not establish pregnancy (p0.05). A lesser average number of 41± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then submitted to RNA sequencing for transcriptome analysis. Using weighted gene co-expression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology (GO) analysis of these clusters revealed enriched biological processes in the coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total- and ICM-cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.

SbWRKY75- and SbWRKY41-mediated jasmonic acid signaling regulates baicalin biosynthesis.

Scutellaria baicalensis Georgi is a traditional Chinese medicinal plant with broad pharmacological activities whose main active ingredient is the flavonoid baicalin. Given its medicinal value and increasing market demand, it is essential to improve the plant's baicalin content. Flavonoid biosynthesis is regulated by several phytohormones, primarily jasmonic acid (JA). In this study, we conducted transcriptome deep sequencing analysis of S. baicalensis roots treated with methyl jasmonate for different durations (1, 3, or 7 hours). Leveraging weighted gene co-expression network analysis and transcriptome data, we identified candidate transcription factor genes involved in the regulation of baicalin biosynthesis. To validate the regulatory interactions, we performed functional assays such as yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays. Our findings demonstrated that SbWRKY75 directly regulates the expression of the flavonoid biosynthetic gene SbCLL-7, whereas SbWRKY41 directly regulates the expression of two other flavonoid biosynthetic genes, SbF6H and SbUGT, thus regulating baicalin biosynthesis. We also obtained transgenic S.baicalensis plants by somatic embryo induction and determined that overexpressing SbWRKY75 increased baicalin content by 14%, while RNAi reduced it by 22%. Notably, SbWRKY41 indirectly regulated baicalin biosynthesis by modulating the expression of SbMYC2.1, SbJAZ3 and SbWRKY75. This study provides valuable insights into the molecular mechanisms underlying JA-mediated baicalin biosynthesis in S. baicalensis. Our results highlight the specific roles of transcription factors, namely SbWRKY75 and SbWRKY41, in the regulation of key biosynthetic genes. Understanding these regulatory mechanisms holds significant potential for developing targeted strategies to enhance baicalin content in S. baicalensis through genetic interventions.

Transcriptome Analysis of Embryogenic and Non-Embryogenic Callus of Picea Mongolica.

Picea mongolica is a rare tree species in China, which is of great significance in combating desertification and improving the harsh ecological environment. Due to the low rate of natural regeneration, high mortality, and susceptibility to pests and cold springs, Picea mongolica has gradually become extinct. At present, somatic embryogenesis (SE) is the most effective method of micro-proliferation in conifers, but the induction rate of embryogenic callus (EC) is low, and EC is difficult to differentiate from non-embryonic callus (NEC). Therefore, the EC and NEC of Picea mongolica were compared from the morphology, histological, physiological, and transcriptional levels, respectively. Morphological observation showed that the EC was white and transparent filamentous, while the NEC was compact and brownish-brown lumpy. Histological analyses showed that the NEC cells were large and loosely arranged; the nuclei attached to the edge of the cells were small; the cytoplasm was low; and the cell gap was large and irregular. In the EC, small cells, closely arranged cells, and a large nucleus and nucleolus were observed. Physiological studies showed significant differences in ROS-scavenging enzymes between the EC and NEC. Transcriptome profiling revealed that 13,267 differentially expressed genes (DEGs) were identified, 3682 were up-regulated, and 9585 were down-regulated. In total, 63 GO terms had significant enrichment, 32 DEGs in plant hormone signal transduction pathway were identified, and 502 different transcription factors (TFs) were characterized into 38 TF families. Meanwhile, we identified significant gene expression trends associated with somatic embryo development in plant hormones (AUX/IAA, YUCCA, LEA, etc.), stress (GST, HSP, GLP, etc.), phenylpropanoid metabolism (4CL, HCT, PAL, etc.), and transcription factors (AP2/ERF, MYB, WOX, etc.). In addition, nine genes were chosen for RT-qPCR, and the results were consistent with RNA-Seq data. This study revealed the changes in morphology, histology, physiology, and gene expression in the differentiation of NEC into EC and laid the foundation for finding the key genes to promote EC formation.

Colchicine-sensitivity Test in Cassava Leaf Lobes and its Effect on Callus and Somatic Embryo Formation

Background: Colchicine acts as a polyploidy inducer but at high concentrations, it causes high cell mortality. To improve the efficiency of colchicine polyploidization in cassava, leaf lobes colchicine-sensitivity tests were carried out and LD50 determined at 0.00, 0.05, 0.10, 0.20 and 0.25g/l colchicine concentrations in the varieties Ankrah, Dagati, Tomfa and Tuaka. Methods: Colchicine treated leaf lobes were regenerated into callus on either 8 mg/l 2, 4-D or Picloram. The calli were subsequently regenerated into somatic embryos by NAA. LD50 of 0.09, 0.11, 0.13 and 0.09 mg/L colchicine concentration were determined for Ankrah, Dagati, Tomfa and Tuaka respectively in 2, 4-D. Similarly, LD50 of 0.12, 0.10, 0.14 and 0.10 mg/L were respectively obtained in Picloram. Result: In 2, 4-D, Ankrah and Tuaka were more sensitive to colchicine than Dagati and Tomfa whereas in picloram, Dagati and Tuaka showed more sensitivity. Callus proliferation differed significantly among varieties and influenced by the concentration of colchicine.

H3K9 methylation patterns during somatic embryogenic competence expression in tamarillo (Solanum betaceum Cav.)

The capacity to regenerate is intrinsic to plants and is the basis of natural asexual propagation and artificial cloning. Despite there are different ways of plant regeneration, they all require a change in cell fate and pluripotency reacquisition, in particular somatic embryogenesis. The mechanisms underlying somatic cell reprogramming for embryogenic competence acquisition, expression and maintenance remain not fully understood. These complex processes have been often associated with epigenetic markers, mainly DNA methylation, while little is known about the possible role of histone modifications. In the present study, the dynamics of global levels and distribution patterns of histone H3 methylation at lysine 9 (H3K9), a major repressive histone modification, were analyzed in somatic embryogenesis-induced cell lines with different embryogenic capacities and during somatic embryo initiation, in the woody species Solanum betaceum. Quantification of global H3K9 methylation showed similar levels in the three types of proliferating calli (embryogenic, long-term and non-embryogenic), kept in high sucrose and auxin-containing medium. Microscopic analyzes revealed heterogeneous cell organization and different cell types, particularly evident in embryogenic callus. The H3K9 dimethylation (H3K9me2) immunofluorescence signal was lower in nuclei of cells showing embryogenic-like and proliferating features, while labeling was higher in vacuolated, non-embryogenic cells with higher proliferation rates. By auxin removal, somatic embryo development was promoted in the embryogenic cell line. During the initiation of this process, increasing levels of global H3K9 methylation were found, together with increasing H3K9me2 immunofluorescence signals, especially in cells of the developing embryo. These results suggest that H3K9 methylation is involved in somatic embryo development, a developmental pathway in which this epigenetic mark could play a role in the gene transcription variation that is associated with embryogenic competence expression in S. betaceum. Altogether, these data provide new insights into the role of this epigenetic mark in somatic embryogenesis in trees, where scarce information is available.

Plant-Derived Smoke and Karrikin 1 in Seed Priming and Seed Biotechnology.

Plant-derived smoke and smoke water (SW) can stimulate seed germination in numerous plants from fire-prone and fire-free areas, including cultivated plants and agricultural weeds. Smoke contains thousands of compounds; only several stimulants and inhibitors have been isolated from smoke. Among the six karrikins present in smoke, karrikin 1 (KAR1) seems to be key for the stimulating effect of smoke. The discovery and activity of highly diluted SW and KAR1 at extremely low concentrations (even at ca. 10-9 M) inducing seed germination of a wide array of horticultural and agricultural plants have created tremendous opportunities for the use of these factors in pre-sowing seed treatment through smoke- or KAR1-priming. This review presents examples of effects exerted by the two types of priming on seed germination and seedling emergence, growth, and development, as well as on the content of some compounds and enzyme activity. Seed biotechnology may involve both SW and KAR1. Some examples demonstrate that SW and/or KAR1 increased the efficiency of somatic embryogenesis, somatic embryo germination and conversion to plantlets. It is also possible to stimulate in vitro seed germination by SW, which allows to use in orchid propagation.

The Impact of Gold Nanoparticles on Somatic Embryogenesis Using the Example of Arabidopsis thaliana.

Although the influence of nanoparticles (NPs) on developmental processes is better understood, little is known about their impact on somatic embryogenesis (SE). This process involves changes in the direction of cell differentiation. Thus, studying the effect of NPs on SE is essential to reveal their impact on cell fate. This study aimed to examine the influence of gold nanoparticles (Au NPs) with different surface charges on the SE of 35S:BBM Arabidopsis thaliana, with particular emphasis on the spatiotemporal localization of pectic arabinogalactan proteins (AGPs) and extensin epitopes in cells changing the direction of their differentiation. The results show that under the influence of nanoparticles, the explant cells of 35S:BBM Arabidopsis thaliana seedling origin did not enter the path of SE. Bulges and the formation of organ-like structures were observed in these explants, in contrast to the control, where somatic embryos developed. Additionally, spatiotemporal changes in the chemical composition of the cell walls during the culture were observed. Under the influence of Au NPs, the following effects were observed: (1) explant cells did not enter the SE pathway, (2) the impacts of Au NPs with different surface charges on the explants were variable, and (3) the compositions of the analyzed pectic AGPs and extensin epitopes were diverse in the cells with different developmental programs: SE (control) and non-SE (treated with Au NPs).

Comparative Transcriptome Analysis between Embryogenic and Non-Embryogenic Callus of Davidia involucrata

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata.

Embryonic abnormalities and genotoxicity induced by 2,4-dichlorophenoxyacetic acid during indirect somatic embryogenesis in Coffea

Indirect somatic embryogenesis (ISE) is a morphogenetic pathway in which somatic cells form callus and, later, somatic embryos (SE). 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin that promotes the proliferation and dedifferentiation of somatic cells, inducing the ISE. However, 2,4-D can cause genetic, epigenetic, physiological and morphological disorders, preventing the regeneration and/or resulting abnormal somatic embryos (ASE). We aimed to evaluate the toxic 2,4-D effect during the Coffea arabica and C. canephora ISE, assessing the SE morphology, global 5-methylcytosine levels (5-mC%) and DNA damage. Leaf explants were inoculated in media with different 2,4-D concentrations. After 90 days, the friable calli were transferred to the regeneration medium, and the number of normal and abnormal SE was monthly counted. The increase of the 2,4-D concentration increased the number of responsive explants in both Coffea. At 9.06, 18.08 and 36.24 μM 2,4-D, C. arabica presented the highest values of responsive explants, differing from C. canephora. Normal and abnormal SE regeneration increased in relation to the time and 2,4-D concentration. Global 5-mC% varied at different stages of the ISE in both Coffea. Furthermore, the 2,4-D concentration positively correlated with global 5-mC%, and with the mean number of ASE. All ASE of C. arabica and C. canephora exhibited DNA damage and showed higher global 5-mC%. The allotetraploid C. arabica exhibited greater tolerance to the toxic effect of 2,4-D than the diploid C. canephora. We conclude that synthetic 2,4-D auxin promotes genotoxic and phytotoxic disorders and promotes epigenetic changes during Coffea ISE.

New methods for sorghum transformation in temperate climates.

Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens-mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach.

Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology with great potential in the reproduction of different species of zootechnical interest, including sheep. This study aimed to carry out a bibliometric analysis of scientific papers published on the application of SCNT in sheep reproduction during the period 1997-2023. The search involved the Science Citation Index Expanded and Social Sciences Citation Index databases of the main collection of the Web of Sciences with different descriptors. A total of 124 scientific papers were analyzed for different bibliometric indicators using the VOSviewer software. Since 2001, the number of SCNT-related papers that have been published concerning sheep reproduction has increased and it has fluctuated in ensuing years. The main authors, research groups, institutions, countries, papers, and journals with the highest number of papers related to the application of SCNT in sheep reproduction were identified, as well as the topics that address the research papers according to the terms: somatic cell, embryo, oocyte, gene expression, SCNT, and sheep.

Somatic Embryo (SE) Formation from Culturing Floral Explants of Rubber Tree (Hevea brasiliensis Muell. Arg.) and Assessment of Genetic Stability by RAPD and SSR Markers

Rubber tree is economically important rubber producing plant of Thailand. At present, a rubber tree plantation is susceptible to white root disease. Therefore, the use of rootstock from early introduce clone that proved to be resistant to white root disease could help sustain growing of rubber tree. Thus, the objectives of this research were to study the effects of plant growth regulators and different explants on somatic embryo (SE) induction of this rubber clone and assessment genetic stability. The results revealed that mix flower explant cultured on MS medium supplemented with 2.0 mg/L 6-benzyladenine (BA) and 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) provided callus subsequent somatic embryo (SE) formation at the highest frequency of 39.84 % and number of cotyledonary embryos (CEs) at 3.25 embryos /callus after 3 passages of subculture in the same culture medium (4 weeks/passage). SEs germinated into embryonic axis at 50 % and further development into shoot at 25 % after transfer to 0.25 mg/L GA3 containing MS medium with the best concentrations of BA and 2,4-D for 4 weeks. The assessment gene stability by RAPD and SSR markers showed no variation between mother plant and in vitro plantlets. In this work, a novel explant source - the floral section of the rubber tree - was used for the first time in order to design an effective technique for in vitro somatic embryogenesis. HIGHLIGHTS The first report on plantlets regeneration using floral explant of rubber tree This mother plant is proved to be resistance to Phytopthora leaf fall and white root diseases Assessment genetic stability of regenerated plantlets by SSR and RAPD markers GRAPHICAL ABSTRACT