Somatic Embryo Induction Research Articles

Impact of activated charcoal on the progression of somatic embryogenesis in grape cells: Histological and transcriptomic perspectives

Somatic embryogenesis is a crucial genetic transformation method in plants. Despite numerous studies being conducted for acquiring embryogenic competence in plant cells, somatic embryo (SE) induction from embryogenic cells (ECs) has attracted relatively limited research attention. In grapes, somatic cell induction is impeded by unidentified inhibitors, which hinders the progression of globular embryo (GE) formation and subsequently embryo development. We found that addition of activated charcoal (AC) to the grape SE induction medium augmented the speed, consistency, and synchronization of embryogenesis. Cell samples cultured in the induction medium with and without AC were thoroughly evaluated through histological anatomy analysis, subcellular structure observation, and comparative transcriptomic analysis. The histological observation revealed the specific timeline of SE formation. An intermediate developmental state, referred to as the globular embryo precursor (PGE), was observed during the induction of GE formation from EC. Members of transcription factor families such as WRKY, MYB, MADS, AP2/ERF, HB-KNOX, HB-HD-ZIP, HB-WOX, NAC, and GRF were identified in the early and middle stages of GE formation. Additionally, we explored the key roles of reactive oxygen species (ROS)-, starch-, cell wall-, and hormone-related genes. The cell wall-related genes were highly enriched in differentially expressed gene sets. Several amyloplasts in EC disappeared, potentially because of hydrolysis, and numerous mitochondria were observed. In AC-added samples, starch was consumed more, and cellulose hydrolysis-related genes were downregulated, whereas cellulose synthesis-related genes had higher expression than AC-free samples. ROS response-related genes were induced when GE induction was initiated. When applied, an optimal concentration of external H2O2 promoted SE initiation in grapes, whereas a high concentration caused delayed GE formation. This study suggests that, by absorbing and releasing substances, the added AC can create a stable buffer environment for cell survival, protect against excessive ROS- or inhibitor-induced cell damage, and support uninterrupted progress of somatic embryogenesis.

Comparative growth response of three rubber (Hevea brasiliensis Muell. Arg) clones in somatic embryo propagation

Abstract Plants have the ability to grow with different responses depending on the clones and varieties of plants. In rubber plants, each clone has specific characteristics or traits that distinguish it from other clones, whether in terms of growth, latex production, or resistance to pests and diseases. The objective of this experiment was to assess the response of three rubber (Hevea brasiliensis Muell. Arg) clones to the same media conditions for the growth and development of somatic embryos (SE). Integuments from three rubber clones, namely PB 260, PB 330, and PB 340, were used as explants to induce embryonic callus on MH media. After four weeks post-induction, the callus was sub-cultured to expressed SE then SE was transferred to the RITA® bio-reactor for plantlets development in liquid MD media. Parameters for each steps, such as the percentage of contamination, percentage of callus response, color of callus, percentage of SE induction, number of stage of SE, and number of shoots were recorded. The results revealed that the highest percentage of viable callus was observed in the PB 260 clone at 23.20%. On SE expression, the highest being in clone PB 340 at 2.04%. On SE development the highest shoot growth was observed in the PB 260 clone, with a total of 5 shoots.

Induction of Somatic Embryogenesis of Pontianak Siamese Orange Cotyledon Cultures on Murashige Skoog Media with the Addition of 2,4-D and Kinetin

Conventional citrus propagation has weaknesses, one of which is susceptible to CVPD(Citrus Vein Phloem Degeneration) and other diseases which are the main cause of the decline in population and production of pontianak siam orange plants in West Kalimantan. One alternative to in vitro propagation of citrus seedlings is the induction of somatic embryogenesis. The purpose of the study was to obtain the best combination of 2,4-D and kinetin concentrations for callus induction and somatic embryo induction from Pontianak siam orange cotyledon explants, observing the developmental phase (stage) of embryos formed. The implementation of the study used a factorial completely randomized design (CRD) consisting of 2 factors. The first factor is the concentration of 2,4-D consisting of 4 concentration levels, namely 0; 0.5; 1; 1.5; 2 mg/L. The second factor is kinetin concentration consisting of 4 concentration levels, namely 0; 0.5; 1; 1.5; 2 mg/L.The observation parameters include the time of callus emergence (hst), the percentage of explants forming callus (%), the texture and color of the callus, observing the stage of the embryo found. The results showed that 72% of treatments were able to induce callus from pontianak siam orange cotyledons with an average callus appearance at 7 hst. The callus formed is compact and crumbly with varied callus colors, namely white, yellowish, greenish and brownish. The best treatment that produces embryogenic callus is the combination of D0.5K0.5; D1K1; D0.5K1.5; D1.5K1.5 and D2K2 with the characteristics of crumbly callus and yellowish and brownish colors. The results of microscopic observations found that the somatic embryo phase formed was the pro-embryo phase.

Exploring somatic embryogenesis in Ajuga multiflora Bunge: Profiling lipophilic metabolites via HPLC, GC-FID, and GCMS– analysis

The present study aims to assess the effect of different concentrations of auxins and combinations of plant growth regulators (PGRs), specifically auxin and cytokinin, on somatic embryogenesis in Ajuga multiflora. We also aim to compare the lipophilic metabolite profiles, such as carotenoids (CAs), fatty acids (FAs), phytosterols (PSs), and tocopherols (TPs), in globular somatic embryos (SEs), germinated SEs, and shoots of SEs derived plantlets. This assessment is carried out utilizing high performance liquid chromatography, gas chromatography-flame-ionization detection, and gas chromatography–mass spectrometry techniques. The results reveal that the application of 2,4-dichlorophenoxyacetic acid (2,4-D) had the most significant impact on somatic embryogenesis, inducing both SE initiation (34.4%) and the number of SEs (3.5) per explant. Among the explants used, leaf explants were found to be the most effective for SE induction (SEI). Additionally, the study explores the combined effect of 2,4-D and cytokinins (N6-benzyladenine, N6-BA, or 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, TDZ) on somatic embryogenesis, highlighting optimal conditions for SEI percentages and the number of SEs per explant. The maximum SE induction rate (100%) and the number of SEs (45.9) per leaf explant were obtained on an SEI medium with 30 µM of 2,4-D and 2.3 µM of TDZ. The maturation and conversion of SEs were also examined, emphasizing the role of GA3 in achieving the highest conversion percentage (94.0 ± 2.7 at 0.8 µM GA3). Furthermore, the study details the biochemical composition during various stages of somatic embryogenesis, including CAs, TPs, PSs, and FAs. Globular SEs displayed comparatively lower concentrations of CAs than their germinated counterparts and shoots from SE-derived plantlets, while the total PSs content remained relatively consistent across all stages. Conversely, fatty acids tended to accumulate more in globular SEs, with the exception of α-Linolenic acid. These findings provide valuable insights into the optimization of somatic embryogenesis protocols in A. multiflora, with implications for plant tissue culture, regeneration and lipophilic metabolites production.

Physiological, Metabolic, and Transcriptomic Analyses Reveal Mechanisms of Proliferation and Somatic Embryogenesis of Litchi (Litchi chinensis Sonn.) Embryogenic Callus Promoted by D-Arginine Treatment.

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H2O2, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism.

Establishment of somatic embryogenesis regeneration system and transcriptome analysis of early somatic embryogenesis in Litchi chinensis

Litchi chinensis Sonn. is an important economic fruit tree in tropical and subtropical regions. Regrettably, the efficiency of plant regeneration via somatic embryogenesis in litchi is typically low due to the poor conversion of embryos to plants. The purpose of this study was to establish a regeneration system via somatic embryogenesis from immature embryos explants in ‘Heiye’ cultivar of litchi. Our results demonstrated that MS medium supplemented with 2.0mg L−1 2,4-D was optimal for callus induction. For somatic embryo (SE) induction, MS medium containing 0.5 g L−1 activated charcoal (AC) was the most effective, while the use of zeatin (ZT) and thidiazuron (TDZ) resulted in abnormal somatic embryos. The rooting and regeneration rate of 2.15% and 17.5%, respectively, were achieved using MS medium supplemented with 0.5 g L−1 AC. Furthermore, transcriptome analysis was performed on embryogenic callus (EC), globular embryo (GE), and heart embryo (HE) to explore the molecular mechanisms of early somatic embryogenesis. 2,587 common DEGs between EC_vs_GE and EC_vs_HE were identified, and the expression patterns of these common DEGs were separated into twelve major clusters. GO annotation and KEGG pathway analysis revealed that these common DEGs were implicated in plant hormone signal transduction, auxin-activated signaling pathway, and other biological processes. Additionally, differentially expression transcription factors were identified, and the function of LcBBM2 which is specifically highly expressed during early somatic embryogenesis was verified. Overexpression of LcBBM2 in tomato promotes callus and shoot formation. Therefore, this study can provide a theoretical basis and technical support for genetic breeding improvement of litchi.

Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

Advancing Hemidesmus indicus propagation and conservation: Somatic embryogenesis, histology, metabolite assessment and genetic stability

Hemidesmus indicus L. R. Br. is a commonly traded ethnomedicinal plant, and a method has been developed to produce somatic embryos (SEs) from calli grown from the leaves of the plant. With a maximum callus induction frequency of 91.66 %, pink-red friable proembryogenic calli were generated on Murashige and Skoog (MS) and 5.0 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) from the leaf explant (LE). With the aim of achieving a high success rate of 95.00 % for the induction of SEs, with a yield of 91.33 SEs per 100 mg of callus, the proembryogenic calli were transferred to fresh culture medium containing MS and 5.0 µM 6-benzylaminopurine (BA). This induction medium allowed the embryos to develop to the torpedo stage, but their further growth into emblings required a different culture medium. ½ MS with 5.0 µM BA and 1.0 µM gibberellic acid (GA3) resulted in 99.16 % maturation and 39.66 emblings regeneration out of 40 immature embryoids, respectively. With a maximum survival rate of 91.67 %, healthy emblings were successfully acclimatized using Soilrite. DNA-based molecular markers (RAPD and ISSR) were used to confirm the genetic similarities of the in vitro grown plants. Histological examinations were performed to determine the ontogeny, morphology, and development of somatic embryoids. Gas chromatography and mass spectrometry (GCMS–) were performed for both the mother and the in vitro generated plant to analyse the root for secondary metabolites. In the physiological and biochemical studies, enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) were found to increase in vitro derived H. indicus plants during different acclimatization periods (0, 7, 14, 21 and 28 days). With increasing acclimatization time, net photosynthetic rate (PN) and total chlorophyll show a decrease at first, then an increase. The protocol provides high callus induction rates, efficient somatic embryogenesis, successful maturation and regeneration, robust acclimatization, genetic fidelity, detailed insight into development, and screening of secondary metabolites.

A WRI1-dependent module is essential for the accumulation of auxin and lipid in somatic embryogenesis of Arabidopsis thaliana.

The potential for totipotency exists in all plant cells; however, the underlying mechanisms remain largely unknown. Earlier findings have revealed that the overexpression of LEAFY COTYLEDON 2 (LEC2) can directly trigger the formation of somatic embryos on the cotyledons of Arabidopsis. Furthermore, cotyledon cells that overexpress LEC2 accumulate significant lipid reserves typically found in seeds. The precise mechanisms and functions governing lipid accumulation in this process remain unexplored. In this study, we demonstrate that WRINKLED1 (WRI1), the key regulator of lipid biosynthesis, is essential for somatic embryo formation, suggesting that WRI1-mediated lipid biosynthesis plays a crucial role in the transition from vegetative to embryonic development. Our findings indicate a direct interaction between WRI1 and LEC2, which enhances the enrichment of LEC2 at downstream target genes and stimulates their induction. Besides, our data suggest that WRI1 forms a complex with LEC1, LEC2, and FUSCA3 (FUS3) to facilitate the accumulation of auxin and lipid for the somatic embryo induction, through strengthening the activation of YUCCA4 (YUC4) and OLEOSIN3 (OLE3) genes. Our results uncover a regulatory module controlled by WRI1, crucial for somatic embryogenesis. These findings provide valuable insights into our understanding of plant cell totipotency.

Development of an Agrobacterium tumefaciens-mediate transformation system for somatic embryos and transcriptome analysis of LcMYB1’s inhibitory effect on somatic embryogenesis in Litchi chinensis.1

Litchi holds paramount economic significance as a global fruit crop. However, the advancement of litchi functional genomics encounters substantial obstacles due to its recalcitrance to stable transformation. Here, we present an efficacious A. tumefaciens-mediated transformation system in somatic embryo of ‘Heiye’ litchi. This system was developed through meticulous optimization of variables encompassing explant selection, A. tumefaciens strain delineation, bacterium concentration, infection duration, and infection methodology. The subsequent validation of the transformation technique in litchi was realized through the ectopic expression of LcMYB1, resulting in the generation of transgenic calli. However, it was discerned that the differentiation of transgenic calli into somatic embryos encountered substantial challenges. To delve into the intricate molecular underpinnings of LcMYB1’s inhibitory role in somatic embryo induction, a comprehensive transcriptome analysis was conducted encompassing embryogenic calli (C), globular embryos (G), and transgenic calli (TC). A total of 1166 common differentially expressed genes (DEGs) were identified between C-vs-G and C-vs-TC. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these common DEGs were most related to plant hormone signal transduction pathways. Furthermore, RT-qPCR corroborated pronounced down-regulation of numerous genes intricately associated with somatic embryos induction within the transgenic calli. The development of this transformation system has provided valuable support for functional genomics research in litchi.

Effect of Picloram and Desiccation on the Somatic Embryogenesis of Lycium barbarum L.

An efficient and reproducible in vitro method for indirect somatic embryogenesis was optimized by culturing leaf and leaf with petiole explants of Lycium barbarum L. Murashige and Skoog (MS) medium, supplemented with various concentrations of Picloram and 2,4-Dichlorophenoxyacetic acid (2,4-D), individually and in combinations, were tested. Picloram (1.0 µM) showed a better response compared to 2,4-D and results indicate it to be a better auxin for induction of somatic embryos for Goji berry. It was seen that the leaf explants were more responsive in callus and somatic embryo induction than the leaf with petiole explant when incubated in the dark for 5 weeks. Embryogenic callus, after being transferred to MS medium containing Benzyl amino purine (BAP) in 1.0 µM, 5.0 µM and 10.0 µM, began to differentiate in light after one week. MS medium with 1.0 µM Picloram + 10 µM BAP resulted as the most favorable treatment for somatic embryogenesis in Lycium barbarum L. Removal of plant growth regulators from MS medium and culturing induced calluses under 16 h photoperiod resulted in globular, heart, torpedo, cotyledons, and further development into plantlets. Well-developed plants have been obtained and are capable of acclimatizing in ex vitro conditions. In addition, the effects of desiccation treatments (0, 1, 3, 6, 9 h, and 12 h) on embryogenic callus for somatic embryo induction were found to be directly proportional to the length of desiccation treatment at room temperature. After 9 h and 12 h of desiccation treatments, 60% and 90% of plated calluses resulted in somatic embryos, respectively. In a L. barbarum callus mass, Acetocarmine and Evans blue double staining differentiated between embryogenic and non-embryogenic callus. These findings will help Goji berry improvement by elite clone production, ex situ conservation projects, scaling up plant production, and agronomy for the commercial production of this superfruit in the future.

Effect of Arabinogalactans on Induction of White-Opaque Somatic Embryos of Avocado (Persea americana Mill.) cv. Duke-7.

The development of somatic embryogenesis in avocado (Persea americana Mill.) has been hampered by different chronic problems. One such problem is the low level of induction of white-opaque somatic embryos (WOSEs) during the process of obtaining full avocado plants. We detected the induction of multiple WOSEs promoted after the placement of three or four small WOSEs over the embryogenic callus of Duke-7. Among the other possible chemical inductors of the Arabinogalactans (AGPs), we identified a family of extracellular plant proteoglycans implicated in many aspects of the in vitro induction of somatic embryos (SE). We extracted AGPs directly from embryogenic cultures of avocado. When the induction/proliferation medium of embryogenic avocado calli (MS-0.1 mg L-1 Picloram) was supplemented with 1-2 mg L-1 AGP, the induction rate of good-quality WOSEs from the embryogenic callus increased significantly (more than ten times that of the control without AGP) and this effect persisted for at least five subcultures after the initial treatment with AGP. AGP also modified the texture and quality of the callus. The effect of AGP extends to other cultivars and proliferation media. Our objectives were to improve the induction of WOSEs and study the effect of AGP in the somatic embryogenesis of avocado.

Molecular mechanism of somatic embryogenesis in paeonia ostii ‘Fengdan’ based on transcriptome analysis combined histomorphological observation and metabolite determination

BackgroundTree peony (Paeonia sect. Moutan DC.) is a famous flower native to China with high ornamental, medicinal, and oil value. However, the low regeneration rate of callus is one of the main constraints for the establishment of a genetic transformation system in tree peony. By histomorphological observation, transcriptomic analysis and metabolite determination, we investigated the molecular mechanism of somatic embryogenesis after the establishment of a culture system and the induction of somatic embryo(SE) formation.ResultsWe found that SE formation was successfully induced when cotyledons were used as explants. A total of 3185 differentially expressed genes were screened by comparative transcriptomic analysis of embryogenic callus (EC), SE, and non-embryogenic callus (NEC). Compared to NEC, the auxin synthesis-related genes GH3.6 and PCO2 were up-regulated, whereas cytokinin dehydrogenase (CKX6) and CYP450 family genes were down-regulated in somatic embryogenesis. In SE, the auxin content was significantly higher than the cytokinin content. The methyltransferase-related gene S-adenosylmethionine synthase (SAMS) and the flavonoid biosynthesis-related gene (ANS and F3’5’H) were down-regulated in somatic embryogenesis. The determination of flavonoids showed that rhoifolin and hyperoside had the highest content in SE. The results of transcriptome analysis were consistent with the relative expression of 8 candidate genes by quantitative polymerase chain reaction analysis.ConclusionThe results revealed that auxin and cytokinin may play a key role in ‘Fengdan’ somatic embryogenesis. The genes related to somatic embryogenesis were revealed, which has partly elucidated the molecular mechanism of somatic embryogenesis in ‘Fengdan’.

Epigenetic modifications and miRNAs determine the transition of somatic cells into somatic embryos.

This review discusses the epigenetic changes during somatic embryo (SE) development, highlights the genes and miRNAs involved in the transition of somatic cells into SEs as a result of epigenetic changes, and draws insights on biotechnological opportunities to study SE development. Somatic embryogenesis from somatic cells occurs in a series of steps. The transition of somatic cells into somatic embryos (SEs) is the most critical step under genetic and epigenetic regulations. Major regulatory genes such as SERK, WUS, BBM, FUS3/FUSA3, AGL15, and PKL, control SE steps and development by turning on and off other regulatory genes. Gene transcription profiles of somatic cells during SE development is the result of epigenetic changes, such as DNA and histone protein modifications, that control and decide the fate of SE formation. Depending on the type of somatic cells and the treatment with plant growth regulators, epigenetic changes take place dynamically. Either hypermethylation or hypomethylation of SE-related genes promotes the transition of somatic cells. For example, the reduced levels of DNA methylation of SERK and WUS promotes SE initiation. Histone modifications also promote SE induction by regulating SE-related genes in somatic cells. In addition, miRNAs contribute to the various stages of SE by regulating the expression of auxin signaling pathway genes (TIR1, AFB2, ARF6, and ARF8), transcription factors (CUC1 and CUC2), and growth-regulating factors (GRFs) involved in SE formation. These epigenetic and miRNA functions are unique and have the potential to regenerate bipolar structures from somatic cells when a pluripotent state is induced. However, an integrated overview of the key regulators involved in SE development and downstream processes is lacking. Therefore, this review discusses epigenetic modifications involved in SE development, SE-related genes and miRNAs associated with epigenetics, and common cis-regulatory elements in the promoters of SE-related genes. Finally, we highlight future biotechnological opportunities to alter epigenetic pathways using the genome editing tool and to study the transition mechanism of somatic cells.

Fungal Elicitation Enhances Vincristine and Vinblastine Yield in the Embryogenic Tissues of Catharanthus roseus.

Fungal elicitation could improve the secondary metabolite contents of in vitro cultures. Herein, we report the effect of Fusarium oxysporum on vinblastine and vincristine alkaloid yields in Catharanthus roseus embryos. The study revealed increased yields of vinblastine and vincristine in Catharanthus tissues. Different concentrations, i.e., 0.05% (T1), 0.15% (T2), 0.25% (T3), and 0.35% (T4), of an F. oxysporum extract were applied to a solid MS medium in addition to a control (T0). Embryogenic calli were formed from the hypocotyl explants of germinating seedlings, and the tissues were exposed to Fusarium extract elicitation. The administration of the F. oxysporum extract improved the growth of the callus biomass, which later differentiated into embryos, and the maximum induction of somatic embryos was noted T2 concentration (102.69/callus mass). A biochemical analysis revealed extra accumulations of sugar, protein, and proline in the fungus-elicitated cultivating tissues. The somatic embryos germinated into plantlets on full-strength MS medium supplemented with 2.24 µM of BA. The germination rate of the embryos and the shoot and root lengths of the embryos were high at low doses of the Fusarium treatment. The yields of vinblastine and vincristine were measured in different treated tissues via high-pressure thin-layer chromatography (HPTLC). The yield of vinblastine was high in mature (45-day old) embryos (1.229 µg g-1 dry weight), which were further enriched (1.267 µg g-1 dry weight) via the F. oxysporum-elicitated treatment, especially at the T2 concentration. Compared to vinblastine, the vincristine content was low, with a maximum of 0.307 µg g-1 dry weight following the addition of the F. oxysporum treatment. The highest and increased yields of vinblastine and vincristine, 7.88 and 15.50%, were noted in F. oxysporum-amended tissues. The maturated and germinating somatic embryos had high levels of SOD activity, and upon the addition of the fungal extracts, the enzyme's activity was further elevated, indicating that the tissues experienced cellular stress which yielded increased levels of vinblastine and vincristine following the T2/T1 treatments. The improvement in the yields of these alkaloids could augment cancer healthcare treatments, making them easy, accessible, and inexpensive.

Somatic Embryogenesis and Plant Regeneration from Stem Explants of Pomegranate

Plant regeneration through somatic embryogenesis provides a solution for maintaining and genetically improving crop or fruit varieties with desirable agronomic traits. For the fruit tree pomegranate (Punica granatum L.), despite some successful applications, the existing somatic embryogenesis protocols are limited by low availability of explants and susceptibility to browning. To address these problems, in this study, we developed an effective system for induction of high-vigor pomegranate somatic embryos derived from stem explants. The usage of stem explants breaks through the difficulty in obtaining material, thus making our system suitable for widespread commercial production. To enhance the performance of our system, we identified the optimal explants, subculture cycles and combination of basal media and plant growth regulators for each step. The results showed that inoculating stem explants onto a Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L 1-naphthaleneacetic acid (NAA) achieved the best induction rate and growth status of pomegranate calli (induction rate = ~72%), and MS medium containing 0.5 mg/L 6-BA and 1.0 mg/L NAA was the optimal condition for the induction of embryogenic calli and somatic embryos (induction rate = ~74% and 79%, respectively). The optimal subculture period for embryogenic calli was found to be 30–35 days. Strong roots were then induced in the developed somatic embryo seedlings, which survived and grew well after transplantation to the natural environment, indicating the good vitality of the induced pomegranate somatic embryos. Together, our system provides a solution to mass somatic embryo induction and plant regeneration of pomegranate and lays a foundation for future genetic transformation and bioengineering improvement of pomegranate with favorable agronomic traits.

Novel System for the Production of the Bioactive N-alkylamide ‘Spilanthol’ Through Somatic Embryogenesis in Acmella ciliata Kunth (Cass.)

Acmella ciliata Kunth (Cass.), a medicinally important plant in the family Asteraceae, has high commercial value because of its traditional phytomedicinal uses. The plant contains many phytochemicals like alkyl amides, alkaloids, tannins, saponins and flavonoids accountable for most of its pharmacological applications. The study presented here reports the callus culture and somatic embryogenesis of this plant thereby raising a novel system for the subsequent production of the N-alkyl amide ‘spilanthol’, the valuable secondary metabolite presents in it. Murashige and Skoog (MS) medium supplemented with auxins either alone or in combination with cytokinins were used for the induction and maturation of somatic embryos. MS medium supplemented with 2,4-D (0.5, 1.0 and 2.0 mg.L-1) produced black friable callus whereas, 1.0 mg.L-1 NAA in combination with 0.5 mg.L-1 BA induced white, slightly purple coloured friable callus which on further subculture to fresh medium induced somatic embryos that germinated into plantlets upon transfer to MS basal medium. The mode of regeneration via somatic embryogenesis was confirmed by histological analysis through free-hand sectioning and stereomicroscopic observation. The plantlets raised through somatic embryogenesis after a short hardening period, were found to acclimatise in the field at 83.33% efficiency and exhibited genetic uniformity with 96.6% similarity in the ISSR analysis. HPLC analysis of in vitro raised embryogenic callus showed 239.512 µg.g-1 spilanthol content which was comparatively higher than the mother plants (92.19 µg.g-1). The bioproduction of the N-alkylamide ‘spilanthol’ through embryogenic callus can be extended for the scale-up production of this bioactive compound using bioreactor technology for the formulation of phytodrugs.

Influence of Basal Salts, Sucrose and Plant Growth Regulator Levels on Nucellar Embryogenesis and Plantlet Regeneration in Monoembryonic Mango Varieties

Current study described stage-wise protocols for in vitro propagation of commercially important varieties of mango. Induction of somatic embryos (SE) and plantlet regeneration was obtained using nucellar explants of three superior monoembryonic mango vars.‘Saroli’, ‘Langra’, and ‘Chaunsa’ were cultivated in Khairpur, Pakistan. The immature fruits (2.5-4.0 cm long) were surface disinfected using a 30% sodium hypochlorite (NaOCl) solution. Results revealed that significantly highest direct somatic embryogenesis (93%) was obtained in var. ‘Chaunsa’ under full dark on culture medium comprising of 2.0 mg L-1 N6 2-isopentenyl adenine (2iP), 0.5 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D). Medium consisted of 2iP 4.0 mg L-1, 2,4-D 1.0 mg L-1 induced significantly highest embryogenic callus (91%) using nucellar explants in var.‘Chaunsa’. Significantly highest germination (95%) of SE was achieved in var. ‘Chaunsa’ on the medium comprising microsalts of MS, macrosalts of B5, 2iP 0.1 mg L-1, Kinetin (Kin) 0.5 mg L-1. Highest shoot length (5.1 cm) and root length (4 cm) were obtained in var. ‘Langra’ on the medium consisted of microsalts of MS, macrosalts of B5, 30 g L-1 sucrose, 200 mg L-1 activated charcoal (AC), 0.1 mg L-1 naphthalene acetic acid (NAA), 0.2 mg L-1 benzyl adenine (BA). Stage-wise protocols established for the regeneration of plantlets can be useful for micropropagation of the other mango varieties of the world.

Genome-Wide Association Mapping of Oil Content and Seed-Related Traits in Shea Tree (Vitellaria paradoxa subsp. nilotica) Populations

Shea tree (Vitellaria paradoxa) is an important fruit tree crop because of its oil used for cooking and the industrial manufacture of cosmetics. Despite its essential benefits, quantitative trait loci linked to the economic traits have not yet been studied. In this study, we performed association mapping on a panel of 374 shea tree accessions using 7530 Single-Nucleotide Polymorphisms (SNPs) markers for oil yield and seed-related traits. Twenty-three SNP markers significantly (−log10 (p) = 4.87) associated with kernel oil content, kernel length, width, and weight were identified. The kernel oil content and kernel width had the most significant marker–trait associations (MTAs) on chromosomes 1 and 8, respectively. Sixteen candidate genes identified were linked to early induction of flower buds and somatic embryos, seed growth and development, substrate binding, transport, lipid biosynthesis, metabolic processes during seed germination, and disease resistance and abiotic stress adaptation. The presence of these genes suggests their role in promoting bioactive functions that condition high oil synthesis in shea seeds. This study provides insights into the important marker-linked seed traits and the genes controlling them, useful for molecular breeding for improving oil yield in the species.

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.