Genome-Wide Identification and Expression Analysis of GATA Family Genes in Dimocarpus longan Lour.

GATA transcription factors, which are DNA-binding proteins with type IV zinc finger binding domains, have a role in transcriptional regulation in biological organisms. They have an indispensable role in the growth and development of plants, as well as in improvements in their ability to face various environmental stresses. To date, GATAs have been identified in many gene families, but the GATA gene in longan (Dimocarpus longan Lour) has not been studied in previous explorations. Various aspects of genes in the longan GATA family, including their identification and classification, the distribution of their positions on chromosomes, their exon/intron structures, a synteny analysis, their expression at different temperatures, concentration of PEG, early developmental stages of somatic embryos and their expression levels in different tissues, and concentrations of exogenous hormones, were investigated in this study. This study showed that the 22 DlGATAs could be divided into four subfamilies. There were 10 pairs of homologous GATA genes in the synteny analysis of DlGATA and AtGATA. Four segmental replication motifs and one pair of tandem duplication events were present among the DlGATA family members. The cis-acting elements located in promoter regions were also found to be enriched with light-responsive elements, which contained related hormone-responsive elements. In somatic embryos, DlGATA4 is upregulated for expression at the globular embryo (GE) stage. We also found that DlGATA expression was strongly up-regulated in roots and stems. The study demonstrated the expression of DlGATA under hormone (ABA and IAA) treatments in embryogenic callus of longan. Under ABA treatment, DlGATA4 was up-regulated and the other DlGATA genes did not respond significantly. Moreover, as demonstrated with qRT-PCR, the expression of DlGATA genes showed strong up-regulated expression levels under 100 µmol·L-1 concentration IAA treatment. This experiment further studied these and simulated their possible connections with a drought response mechanism, while correlating them with their expression under PEG treatment. Overall, this experiment explored the GATA genes and dug into their evolution, structure, function, and expression profile, thus providing more information for a more in-depth study of the characteristics of the GATA family of genes.

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.

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

BACKGROUND: Tree 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. RESULTS: We 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. CONCLUSION: The 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'.

Transcriptomic Profiling of Embryogenic and Non-Embryogenic Callus Provides New Insight into the Nature of Recalcitrance in Cannabis.

Differential gene expression profiles of various cannabis calli including non-embryogenic and embryogenic (i.e., rooty and embryonic callus) were examined in this study to enhance our understanding of callus development in cannabis and facilitate the development of improved strategies for plant regeneration and biotechnological applications in this economically valuable crop. A total of 6118 genes displayed significant differential expression, with 1850 genes downregulated and 1873 genes upregulated in embryogenic callus compared to non-embryogenic callus. Notably, 196 phytohormone-related genes exhibited distinctly different expression patterns in the calli types, highlighting the crucial role of plant growth regulator (PGRs) signaling in callus development. Furthermore, 42 classes of transcription factors demonstrated differential expressions among the callus types, suggesting their involvement in the regulation of callus development. The evaluation of epigenetic-related genes revealed the differential expression of 247 genes in all callus types. Notably, histone deacetylases, chromatin remodeling factors, and EMBRYONIC FLOWER 2 emerged as key epigenetic-related genes, displaying upregulation in embryogenic calli compared to non-embryogenic calli. Their upregulation correlated with the repression of embryogenesis-related genes, including LEC2, AGL15, and BBM, presumably inhibiting the transition from embryogenic callus to somatic embryogenesis. These findings underscore the significance of epigenetic regulation in determining the developmental fate of cannabis callus. Generally, our results provide comprehensive insights into gene expression dynamics and molecular mechanisms underlying the development of diverse cannabis calli. The observed repression of auxin-dependent pathway-related genes may contribute to the recalcitrant nature of cannabis, shedding light on the challenges associated with efficient cannabis tissue culture and regeneration protocols.

The Establishment of a Genetic Transformation System and the Acquisition of Transgenic Plants of Oriental Hybrid Lily (Lilium L.).

Lily (Lilium spp.) has elegant flowers and beautiful colors, which makes it popular among people. However, the poor stress resistance and self-propagation ability of lily limit its application in landscaping to a great extent. In addition, transgenic technology is an important means to improve plant characteristics, but the lack of a stable and efficient genetic transformation system is still an important factor restricting the development of lily transgenic technology. Therefore, this study established a good lily regeneration system by screening different explants and plant growth regulators of different concentrations. Then, the genetic transformation system of lily was optimized by screening the critical concentration of antibiotics, the concentration of bacterial solution, and the infection time. Finally, the homologous lily cold resistance gene LlNAC2 and bulblet generation gene LaKNOX1 were successfully transferred to 'Siberia' and 'Sorbonne' to obtain lily transgenic lines. The results showed that when the stem axis was used as explant in 'Siberia', the induction rate was as high as 87%. The induction rate of 'Sorbonne' was as high as 91.7% when the filaments were used as explants. At the same time, in the optimized genetic transformation system, the transformation rate of 'Siberia' and 'Sorbonne' was up to 60%. In conclusion, this study provides the theoretical basis and technical support for improving the resistance and reproductive ability of Oriental lily and the molecular breeding of lily.

Embryogenic Stem Cell Identity after Protoplast Isolation from Daucus carota and Recovery of Regeneration Ability through Protoplast Culture.

Protoplasts are single cells isolated from tissues or organs and are considered a suitable system for cell studies in plants. Embryogenic cells are totipotent stem cells, but their regeneration ability decreases or becomes lost altogether with extension of the culture period. In this study, we isolated and cultured EC-derived protoplasts (EC-pts) from carrots and compared them with non-EC-derived protoplasts (NEC-pts) with respect to their totipotency. The protoplast isolation conditions were optimized, and the EC-pts and NEC-pts were characterized by their cell size and types. Both types of protoplasts were then embedded using the alginate layer (TAL) method, and the resulting EC-pt-TALs and NEC-pt-TALs were cultured for further regeneration. The expression of the EC-specific genes SERK1, WUS, BBM, LEC1, and DRN was analyzed to confirm whether EC identity was maintained after protoplast isolation. The protoplast isolation efficiency for EC-pts was 2.4-fold higher than for NEC-pts (3.5 × 106 protoplasts·g−1 FW). In the EC-pt group, protoplasts < 20 µm accounted for 58% of the total protoplasts, whereas in the NEC-pt group, small protoplasts accounted for only 26%. In protoplast culture, the number of protoplasts that divided was 2.6-fold higher for EC-pts than for NEC-pts (7.7 × 104 protoplasts·g−1 FW), with a high number of plants regenerated for EC-pt-TALs, whereas no plants were induced by NEC-pt-TAL. Five times more plants were regenerated from EC-pts than from ECs. Regarding the expression of EC-specific genes, WUS and SERK1 expression increased 12-fold, and LEC1 and BBM expression increased 3.6−6.4-fold in isolated protoplasts compared with ECs prior to protoplast isolation (control). These results reveal that the protoplast isolation process did not affect the embryogenic cell identity; rather, it increased the plant regeneration rate, confirming that EC-derived protoplast culture may be an efficient system for increasing the regeneration ability of old EC cultures through the elimination of old and inactivate cells. EC-derived protoplasts may also represent an efficient single-cell system for application in new breeding technologies such as genome editing.

Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos.

The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These EC induction traits showed high broad-sense heritability (>80%), and significantly negative correlations were observed between REC and each of the other traits across multiple environments. A total of 41 QTLs for EC induction were identified, among which 13, 12, 10, and 6 QTLs were responsible for DC, RSF, LS, and REC, respectively. Among them, three major QTLs accounted for >10% of the phenotypic variation, including qLS1-1 (11.54%), qLS1-3 (10.68%), and qREC4-1 (11.45%). Based on the expression data of the 215 candidate genes located in these QTL intervals, we performed a weighted gene co-expression network analysis (WGCNA). A combined use of KEGG pathway enrichment and eigengene-based connectivity (KME) values identified the EC induction-associated module and four hub genes (Zm00001d028477, Zm00001d047896, Zm00001d034388, and Zm00001d022542). Gene-based association analyses validated that the variations in Zm00001d028477 and Zm00001d034388, which were involved in tryptophan biosynthesis and metabolism, respectively, significantly affected EC induction ability among different inbred lines. Our study brings novel insights into the genetic and molecular mechanisms of EC induction and helps to promote marker-assisted selection of high-REC varieties in maize.

Glutathione Plays a Positive Role in the Proliferation of Pinus koraiensis Embryogenic Cells.

In the large-scale breeding of conifers, cultivating embryogenic cells with good proliferative capacity is crucial in the process of somatic embryogenesis. In the same cultural environment, the proliferative capacity of different cell lines is significantly different. To reveal the regulatory mechanism of proliferation in woody plant cell lines with different proliferative potential, we used Korean pine cell lines with high proliferative potential 001#-001 (Fast) and low proliferative potential 001#-010 (Slow) for analysis. A total of 17 glutathione-related differentially expressed genes was identified between F and S cell lines. A total of 893 metabolites was obtained from the two cell lines in the metabolomic studies. A total of nine metabolites related to glutathione was significantly upregulated in the F cell line compared with the S cell line. The combined analyses revealed that intracellular glutathione might be the key positive regulator mediating the difference in proliferative capacity between F and S cell lines. The qRT-PCR assay validated 11 differentially expressed genes related to glutathione metabolism. Exogenous glutathione and its synthase inhibitor L-buthionine-sulfoximine treatment assay demonstrated the positive role of glutathione in the proliferation of Korean pine embryogenic cells.

Elevated transcription of transposable elements is accompanied by het-siRNA-driven de novo DNA methylation in grapevine embryogenic callus.

BACKGROUND: Somatic variation is a valuable source of trait diversity in clonally propagated crops. In grapevine, which has been clonally propagated worldwide for centuries, important phenotypes such as white berry colour are the result of genetic changes caused by transposable elements. Additionally, epiallele formation may play a role in determining geo-specific ('terroir') differences in grapes and thus ultimately in wine. This genomic plasticity might be co-opted for crop improvement via somatic embryogenesis, but that depends on a species-specific understanding of the epigenetic regulation of transposable element (TE) expression and silencing in these cultures. For this reason, we used whole-genome bisulphite sequencing, mRNA sequencing and small RNA sequencing to study the epigenetic status and expression of TEs in embryogenic callus, in comparison with leaf tissue. RESULTS: We found that compared with leaf tissue, grapevine embryogenic callus cultures accumulate relatively high genome-wide CHH methylation, particularly across heterochromatic regions. This de novo methylation is associated with an abundance of transcripts from highly replicated TE families, as well as corresponding 24 nt heterochromatic siRNAs. Methylation in the TE-specific CHG context was relatively low over TEs located within genes, and the expression of TE loci within genes was highly correlated with the expression of those genes. CONCLUSIONS: This multi-'omics analysis of grapevine embryogenic callus in comparison with leaf tissues reveals a high level of genome-wide transcription of TEs accompanied by RNA-dependent DNA methylation of these sequences in trans. This provides insight into the genomic conditions underlying somaclonal variation and epiallele formation in plants regenerated from embryogenic cultures, which is an important consideration when using these tissues for plant propagation and genetic improvement.

Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection.

BACKGROUND: The Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation. RESULTS: In this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells. CONCLUSIONS: Our findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.).

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

The regeneration of Acer rubrum L. "October Glory" through embryonic callus.

BACKGROUND: Tissue culture and rapid propagation technology is an important way to solve the difficulties of plant propagation. This experiment aims to explore the appropriate conditions at each stage of the red maple's tissue culture process and to obtain plantlets, thus providing a theoretical basis for the establishment of the red maple's tissue culture system. RESULTS: The results showed that the stem segment is the most suitable explant for inducing embryogenic callus. The MS (Murashige&Skoog) + 0.8 mg/L TDZ (Thidiazuron) + 1.0 mg/L 6-BA (6-Benzylaminopurine) + 0.5 mg/L IAA(Indole-3-acetic acid) + 35 g/L sucrose+ 7.5 g/L semi-fixed medium was the best for callus formation. When selecting type VI callus as embryonic callus induction material, MS + 0.6 mg/L TDZ + 0.5 mg/L 6-BA + 2.0 mg/L IAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium can get embryonic callus. The optimal medium for adventitious bud induction is MS + 1.0 mg/L TDZ + 3.0 mg/L 6-BA+ 0.2 mg/L NAA (1-Naphthaleneacetic acid) + 1.2 mg/L IAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium. The induction rate of adventitious roots in MS + 0.6 mg/L TDZ + 1.0 mg/L 6-BA+ 3 mg/L NAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium was the highest, reaching 76%. CONCLUSIONS: In the course of our research, we found that PGRs play an important role in the callus induction stage, and the effect of TDZ is particularly obvious; The callus cells grow and proliferate according to the "S" growth curve, and can be sub-cultured when the highest growth point is reached to maintain the rapid proliferation of the callus cells and to avoid inactivation of callus caused by tight niche.

Polycomb Repressive Complex 2-mediated histone modification H3K27me3 is associated with embryogenic potential in Norway spruce.

Epigenetic reprogramming during germ cell formation is essential to gain pluripotency and thus embryogenic potential. The histone modification H3K27me3, which is catalysed by the Polycomb repressive complex 2 (PRC2), regulates important developmental processes in both plants and animals, and defects in PRC2 components cause pleiotropic developmental abnormalities. Nevertheless, the role of H3K27me3 in determining embryogenic potential in gymnosperms is still elusive. To address this, we generated H3K27me3 profiles of Norway spruce (Picea abies) embryonic callus and non-embryogenic callus using CUT&RUN, which is a powerful method for chromatin profiling. Here, we show that H3K27me3 mainly accumulated in genic regions in the Norway spruce genome, similarly to what is observed in other plant species. Interestingly, H3K27me3 levels in embryonic callus were much lower than those in the other examined tissues, but markedly increased upon embryo induction. These results show that H3K27me3 levels are associated with the embryogenic potential of a given tissue, and that the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Thus, our study provides novel insights into the role of this epigenetic mark in spruce embryogenesis and reinforces the importance of PRC2 as a key regulator of cell fate determination across different plant species.

Expression of AtLEC2 and AtIPTs promotes embryogenic callus formation and shoot regeneration in tobacco.

BACKGROUND: LEAFY COTYLEDON 2 (LEC2) acts throughout embryo morphogenesis and maturation phase to maintain embryogenic identity. Our previous study stated that Arabidopsis thaliana LEC2 (AtLEC2) driven by glucocorticoid receptor-dexamethasone (GR-DEX) inducible system (AtLEC2-GR) triggers embryogenic callus formation in tobacco (Nicotiana tabacum). RESULTS: In this study, the adenosine phosphate isopentenyltransferase genes AtIPT3, AtIPT7 and the tRNA isopentenyltransferase gene AtIPT9 were overexpressed in the AtLEC2-GR transgenic background. In the AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR seedlings, high-quality embryogenic callus was obtained under the DEX condition, and the shoot regeneration efficiency was 2 to 3.5 folds higher than AtLEC2-GR alone on hormone free medium without DEX. Transcriptome analyses showed that up-regulated BBM, L1L, ABI3, and FUS3 might function during embryogenic callus formation. However, at the shoot regeneration stage, BBM, L1L, ABI3, and FUS3 were down-regulated and Type-B ARRs were up-regulated, which might contribute to the increased shoot regeneration rate. CONCLUSIONS: A novel system for inducing shoot regeneration in tobacco has been developed using the GR-DEX system. Induced expression of AtLEC2 triggers embryogenic callus formation and overexpression of AtIPT7 or AtIPT9 improves shoot regeneration without exogenous cytokinin.

Transcriptome-wide effect of DE-ETIOLATED1 (DET1) suppression in embryogenic callus of Carica papaya.

Papaya is one of the most nutritional fruits, rich in vitamins, carotenoids, flavonoids and other antioxidants. Previous studies showed phytonutrient improvement without affecting quality in tomato fruit and rapeseed through the suppression of DE-ETIOLATED-1 (DET1), a negative regulator in photomorphogenesis. This study is conducted to study the effects of DET1 gene suppression in papaya embryogenic callus. Immature zygotic embryos were transformed with constitutive expression of a hairpin DET1 construct (hpDET1). PCR screening of transformed calli and reverse transcription quantitative PCR (RT-qPCR) verified that DET1 gene downregulation in two of the positive transformants. High-throughput cDNA 3' ends sequencing on DET1-suppressed and control calli for transcriptomic analysis of global gene expression identified a total of 452 significant (FDR < 0.05) differentially expressed genes (DEGs) upon DET1 suppression. The 123 upregulated DEGs were mainly involved in phenylpropanoid biosynthesis and stress responses, compared to 329 downregulated DEGs involved in developmental processes, lipid metabolism, and response to various stimuli. This is the first study to demonstrate transcriptome-wide relationship between light-regulated pathway and secondary metabolite biosynthetic pathways in papaya. This further supports that the manipulation of regulatory gene involved in light-regulated pathway is possible for phytonutrient improvement of tropical fruit crops.

De novo assembly and comparative analysis of the transcriptome of embryogenic callus formation in bread wheat (Triticum aestivum L.).

BACKGROUND: During asexual reproduction the embryogenic callus can differentiate into a new plantlet, offering great potential for fostering in vitro culture efficiency in plants. The immature embryos (IMEs) of wheat (Triticum aestivum L.) are more easily able to generate embryogenic callus than mature embryos (MEs). To understand the molecular process of embryogenic callus formation in wheat, de novo transcriptome sequencing was used to generate transcriptome sequences from calli derived from IMEs and MEs after 3d, 6d, or 15d of culture (DC). RESULTS: In total, 155 million high quality paired-end reads were obtained from the 6 cDNA libraries. Our de novo assembly generated 142,221 unigenes, of which 59,976 (42.17%) were annotated with a significant Blastx against nr, Pfam, Swissprot, KOG, KEGG, GO and COG/KOG databases. Comparative transcriptome analysis indicated that a total of 5194 differentially expressed genes (DEGs) were identified in the comparisons of IME vs. ME at the three stages, including 3181, 2085 and 1468 DEGs at 3, 6 and 15 DC, respectively. Of them, 283 overlapped in all the three comparisons. Furthermore, 4731 DEGs were identified in the comparisons between stages in IMEs and MEs. Functional analysis revealed that 271transcription factor (TF) genes (10 overlapped in all 3 comparisons of IME vs. ME) and 346 somatic embryogenesis related genes (SSEGs; 35 overlapped in all 3 comparisons of IME vs. ME) were differentially expressed in at least one comparison of IME vs. ME. In addition, of the 283 overlapped DEGs in the 3 comparisons of IME vs. ME, excluding the SSEGs and TFs, 39 possessed a higher rate of involvement in biological processes relating to response to stimuli, in multi-organism processes, reproductive processes and reproduction. Furthermore, 7 were simultaneously differentially expressed in the 2 comparisons between the stages in IMEs, but not MEs, suggesting that they may be related to embryogenic callus formation. The expression levels of genes, which were validated by qRT-PCR, showed a high correlation with the RNA-seq value. CONCLUSIONS: This study provides new insights into the role of the transcriptome in embryogenic callus formation in wheat, and will serve as a valuable resource for further studies addressing embryogenic callus formation in plants.

Transgenic papaya: a useful platform for oral vaccines.

MAIN CONCLUSION: Transgenic papaya callus lines expressing the components of the S3Pvac vaccine constitute a stable platform to produce an oral vaccine against cysticercosis caused by Taenia solium or T. crassiceps. The development of effective delivery systems to cope with the reduced immunogenicity of new subunit vaccines is a priority in vaccinology. Herein, experimental evidence supporting a papaya-based platform to produce needle-free, recombinant, highly immunogenic vaccines is shown. Papaya (Carica papaya) callus lines were previously engineered by particle bombardment to express the three protective peptides of the S3Pvac anti-cysticercosis vaccine (KETc7, KETc12, KETc1). Calli were propagated in vitro, and a stable integration and expression of the target genes has been maintained, as confirmed by PCR, qRT-PCR, and HPLC. These results point papaya calli as a suitable platform for long-term transgenic expression of the vaccine peptides. The previously demonstrated protective immunogenic efficacy of S3Pvac-papaya orally administered to mice is herein confirmed in a wider dose-range and formulated with different delivery vehicles, adequate for oral vaccination. This protection is accompanied by an increase in anti-S3Pvac antibody titers and a delayed hypersensitivity response against the vaccine. A significant increase in CD4+ and CD8+ lymphocyte proliferation was induced in vitro by each vaccine peptide in mice immunized with the lowest dose of S3Pvac papaya (0.56 ng of the three peptides in 0.1 µg of papaya callus total protein per mouse). In pigs, the obliged intermediate host for Taenia solium, S3Pvac papaya was also immunogenic when orally administered in a two-log dose range. Vaccinated pigs significantly increased anti-vaccine antibodies and mononuclear cell proliferation. Overall, the oral immunogenicity of this stable S3Pvac-papaya vaccine in mice and pigs, not requiring additional adjuvants, supports the interest in papaya callus as a useful platform for plant-based vaccines.

Gene expression atlas for the food security crop cassava.

Cassava (Manihot esculenta) feeds c. 800 million people world-wide. Although this crop displays high productivity under drought and poor soil conditions, it is susceptible to disease, postharvest deterioration and the roots contain low nutritional content. Here, we provide molecular identities for 11 cassava tissue/organ types through RNA-sequencing and develop an open access, web-based interface for further interrogation of the data. Through this dataset, we consider the physiology of cassava. Specifically, we focus on identification of the transcriptional signatures that define the massive, underground storage roots used as a food source and the favored target tissue for transgene integration and genome editing, friable embryogenic callus (FEC). Further, we identify promoters able to drive strong expression in multiple tissue/organs. The information gained from this study is of value for both conventional and biotechnological improvement programs.

Differential proteome analysis during early somatic embryogenesis in Musa spp. AAA cv. Grand Naine.

KEY MESSAGE: Endogenous hormone secretion proteins along with stress and defense proteins play predominant role in banana embryogenesis. This study reveals the underlying molecular mechanism during transition from vegetative to embryogenic state. Banana (Musa spp.) is well known globally as a food fruit crop for millions. The requirement of quality planting material of banana is enormous. Although mass multiplication through tissue culture is in vogue, high-throughput techniques like somatic embryogenesis (SE) as a mass multiplication tool needs to be improved. Apart from clonal propagation, SE has extensive applications in genetic improvement and mutation. SE in banana is completely genome-dependent and most of the commercial cultivars exhibit recalcitrance. Thus, understanding the molecular basis of embryogenesis in Musa will help to develop strategies for mass production of quality planting material. In this study, differentially expressed proteins between embryogenic calli (EC) and non-embryogenic calli (NEC) with respect to the explant, immature male flower buds (IMFB), of cv. Grand Naine (AAA) were determined using two-dimensional gel electrophoresis (2DE). The 2DE results were validated through qRT-PCR. In total, 65 proteins were identified: 42 were highly expressed and 23 were less expressed in EC compared to NEC and IMFB. qRT-PCR analysis of five candidate proteins, upregulated in EC, were well correlated with expression at transcript level. Further analysis of proteins showed that embryogenesis in banana is associated with the control of oxidative stress. The regulation of ROS scavenging system and protection of protein structure occurred in the presence of heat shock proteins. Alongside, high accumulation of stress-related cationic peroxidase and plant growth hormone-related proteins like indole-3-pyruvate monooxygenase and adenylate isopentenyltransferase in EC revealed the association with the induction of SE.

Genome-wide transcriptome profiling of Carica papaya L. embryogenic callus.

Genome-wide transcriptome profiling is a powerful tool to study global gene expression patterns in plant development. We report the first transcriptome profile analysis of papaya embryogenic callus to improve our understanding on genes associated with somatic embryogenesis. By using 3' mRNA-sequencing, we generated 6,190,687 processed reads and 47.0% were aligned to papaya genome reference, in which 21,170 (75.4%) of 27,082 annotated genes were found to be expressed but only 41% was expressed at functionally high levels. The top 10% of genes with high transcript abundance were significantly enriched in biological processes related to cell proliferation, stress response, and metabolism. Genes functioning in somatic embryogenesis such as SERK and LEA, hormone-related genes, stress-related genes, and genes involved in secondary metabolite biosynthesis pathways were highly expressed. Transcription factors such as NAC, WRKY, MYB, WUSCHEL, Agamous-like MADS-box protein and bHLH important in somatic embryos of other plants species were found to be expressed in papaya embryogenic callus. Abundant expression of enolase and ADH is consistent with proteome study of papaya somatic embryo. Our study highlights that some genes related to secondary metabolite biosynthesis, especially phenylpropanoid biosynthesis, were highly expressed in papaya embryogenic callus, which might have implication for cell factory applications. The discovery of all genes expressed in papaya embryogenic callus provides an important information into early biological processes during the induction of embryogenesis and useful for future research in other plant species.