Monocotyledonous plants graft at the embryonic root-shoot interface.

Grafting is possible in both animals and plants. Although in animals the process requires surgery and is often associated with rejection of non-self, in plants grafting is widespread, and has been used since antiquity for crop improvement1. However, in the monocotyledons, which represent the second largest group of terrestrial plants and include many staple crops, the absence of vascular cambium is thought to preclude grafting2. Here we show that the embryonic hypocotyl allows intra- and inter-specific grafting in all three monocotyledon groups: the commelinids, lilioids and alismatids. We show functional graft unions through histology, application of exogenous fluorescent dyes, complementation assays for movement of endogenous hormones, and growth of plants to maturity. Expression profiling identifies genes that unify the molecular response associated with grafting in monocotyledons and dicotyledons, but also gene families that have not previously been associated with tissue union. Fusion of susceptible wheat scions to oat rootstocks confers resistance to the soil-borne pathogen Gaeumannomyces graminis. Collectively, these data overturn the consensus that monocotyledons cannot form graft unions, and identify the hypocotyl (mesocotyl in grasses) as a meristematic tissue that allows this process. We conclude that graft compatibility is a shared ability among seed-bearing plants.

Microtubule organization changes severely after mannitol and n-butanol treatments inducing microspore embryogenesis in bread wheat.

BACKGROUND: A mannitol stress treatment and a subsequent application of n-butanol, known as a microtubule-disrupting agent, enhance microspore embryogenesis (ME) induction and plant regeneration in bread wheat. To characterize changes in cortical (CMT) and endoplasmic (EMT) microtubules organization and dynamics, associated with ME induction treatments, immunocytochemistry studies complemented by confocal laser scanning microscopy (CLSM) were accomplished. This technique has allowed us to perform advanced 3- and 4D studies of MT architecture. The degree of MT fragmentation was examined by the relative fluorescence intensity quantification. RESULTS: In uni-nucleated mannitol-treated microspores, severe CMT and EMT fragmentation occurs, although a complex network of short EMT bundles protected the nucleus. Additional treatment with n-butanol resulted in further depolymerization of both CMT and EMT, simultaneously with the formation of MT aggregates in the perinuclear region. Some aggregates resembled a preprophase band. In addition, a portion of the microspores progressed to the first mitotic division during the treatments. Bi-nucleate pollen-like structures showed a high MT depolymerization after mannitol treatment and numerous EMT bundles around the vegetative and generative nuclei after n-butanol. Interestingly, bi-nucleate symmetric structures showed prominent stabilization of EMT. CONCLUSIONS: Fragmentation and stabilization of microtubules induced by mannitol- and n-butanol lead to new configurations essential for the induction of microspore embryogenesis in bread wheat. These results provide robust insight into MT dynamics during EM induction and open avenues to address newly targeted treatments to induce ME in recalcitrant species.

Unpollinated Ovaries Used to Produce Doubled Haploid Lines in Durum Wheat.

The use of doubled haploid lines improves the efficiency of cultivar development and homozygous genotypes can be obtained in one generation, as opposed to conventional line production, which requires several cycles of self-pollination. However, in durum wheat (Triticum turgidum subsp. durum Desf.), the low efficiency of green plant regeneration and the very high frequency of albino plants hinder the application of this technique.We observed the success of using gynogenesis for durum wheat and the significant influence of growing conditions on ovary and callus development, and on plant regeneration. Our results suggested that the cold pretreatment for 2 weeks is efficient for durum wheat. Furthermore, the addition of 2,4-D, vitamins and glutamine, and the use of maltose as sugar source in media improved the ovary regeneration. We describe in this work an efficient method to regenerate green plants from in vitro durum wheat gynogenesis .

Development of a cell-free protein synthesis system for practical use.

Conventional cell-free protein synthesis systems had been the major platform to study the mechanism behind translating genetic information into proteins, as proven in the central dogma of molecular biology. Albeit being powerful research tools, most of the in vitro methods at the time failed to produce enough protein for practical use. Tremendous efforts were being made to overcome the limitations of in vitro translation systems, though mostly with limited success. While great knowledge was accumulated on the translation mechanism and ribosome structure, researchers rationalized that it may be impossible to fully reconstitute such a complex molecular process in a test tube. This review will examine how we have solved the difficulties holding back progress. Our newly developed cell-free protein synthesis system is based on wheat embryos and has many excellent characteristics, in addition to its high translation activity and robustness. Combined with other novel elementary technologies, we have established cell-free protein synthesis systems for practical use in research and applied sciences.

The Landscape of the Genomic Distribution and the Expression of the F-Box Genes Unveil Genome Plasticity in Hexaploid Wheat during Grain Development and in Response to Heat and Drought Stress.

FBX proteins are subunits of the SCF complex (Skp1-cullin-FBX) belonging to the E3 ligase family, which is involved in the ubiquitin-proteasome 26S (UPS) pathway responsible for the post-translational protein turnover. By targeting, in a selective manner, key regulatory proteins for ubiquitination and 26S proteasome degradation, FBX proteins play a major role in plant responses to diverse developmental and stress conditions. Although studies on the genomic organization of the FBX gene family in various species have been reported, knowledge related to bread wheat (Triticum aestivum) is scarce and needs to be broadened. Using the latest assembly of the wheat genome, we identified 3670 TaFBX genes distributed non-homogeneously within the three subgenomes (A, B and D) and between the 21 chromosomes, establishing it as one of the richest gene families among plant species. Based on the presence of the five different chromosomal regions previously identified, the present study focused on the genomic distribution of the TaFBX family and the identification of differentially expressed genes during the embryogenesis stages and in response to heat and drought stress. Most of the time, when comparing the expected number of genes (taking into account the formal gene distribution on the entire wheat genome), the TaFBX family harbors a different pattern at the various stratum of observation (subgenome, chromosome, chromosomal regions). We report here that the local gene expansion of the TaFBX family must be the consequence of multiple and complex events, including tandem and small-scale duplications. Regarding the differentially expressed TaFBX genes, while the majority of the genes are localized in the distal chromosomal regions (R1 and R3), differentially expressed genes are more present in the interstitial regions (R2a and R2b) than expected, which could be an indication of the preservation of major genes in those specific chromosomal regions.

A competence of embryo-derived tissues of tetraploid cultivated wheat species Triticum dicoccum and Triticum timopheevii for efficient and stable transgenesis mediated by particle inflow gun.

BACKGROUND: The ability to engineer cereal crops by gene transfer technology is a powerful and informative tool for discovering and studying functions of genes controlling environmental adaptability and nutritional value. Tetraploid wheat species such as emmer wheat and Timopheevi wheat are the oldest cereal crops cultivated in various world areas long before the Christian era. Nowadays, these hulled wheat species are gaining new interest as donors for gene pools responsible for the improved grain yield and quality, tolerance for abiotic and biotic stress, resistance to pests and disease. The establishing of efficient gene transfer techniques for emmer and Timopheevi wheat may help in creation of modern polyploid wheat varieties. RESULTS: In the present study, we describe a robust protocol for the production of fertile transgenic plants of cultivated emmer wheat (Russian cv. 'Runo') using a biolistic delivery of a plasmid encoding the gene of green fluorescent protein (GFP) and an herbicide resistance gene (BAR). Both the origin of target tissues (mature or immature embryos) and the type of morphogenic calli (white or translucent) influenced the efficiency of stable transgenic plant production in emmer wheat. The bombardment of nodular white compact calluses is a major factor allowed to achieve the highest transformation efficiency of emmer wheat (on average, 12.9%) confirmed by fluorescence, PCR, and Southern blot. In the absence of donor plants for isolation of immature embryos, mature embryo-derived calluses could be used as alternative tissues for recovering transgenic emmer plants with a frequency of 2.1%. The biolistic procedure based on the bombardment of immature embryo-derived calluses was also successful for the generation of transgenic Triticum timopheevii wheat plants (transformation efficiency of 0.5%). Most of the primary events transmitted the transgene expression to the sexual progeny. CONCLUSION: The procedures described here can be further used to study the functional biology and contribute to the agronomic improvement of wheat. We also recommend involving in such research the Russian emmer wheat cv. 'Runo', which demonstrates a high capacity for biolistic-mediated transformation, exceeding the previously reported values for different genotypes of polyploid wheat.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects. Moreover, GRF4-GIF1 induced efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. We also combined GRF4-GIF1 with CRISPR-Cas9 genome editing and generated 30 edited wheat plants with disruptions in the gene Q (AP2L-A5). Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus, suggesting that this strategy can be applied to dicot crops.

Trichostatin A and sodium butyrate promotes plant regeneration in common wheat.

Histone acetylation modification plays a vital role in plant cell division and differentiation. However, the function on wheat mature embryo culture has not been reported. Here, we used the mature embryo of wheat genotypes including CB037, Fielder, and Chinese Spring (CS) as materials to analyze the effects of different concentrations of trichostatin A (TSA) and sodium butyrate (SB) on plant regeneration efficiency. The results showed that, compared with the control group, the induction rates of embryogenic callus and green shoot were significantly increased with the addition of 0.5 µM TSA, while they were reduced under treatment of 2.5 µM TSA on wheat mature embryo. With the respective addition of 200 µM and 1000 µM SB, regeneration frequency of three genotypes was enhanced, especially in Fielder, which reached significant difference compared with the control group. Unfortunately, 0.5 µM TSA and 200 µM SB combination had no apparent effect on wheat regeneration frequency. The results indicated that TSA and SB increase plant regeneration in common wheat. In addition, TSA had a common effect and SB had different effect among genotypes on wheat regeneration frequency. The mechanism of action needs further investigation.

Identification and characterization of long non-coding RNAs regulating resistant starch biosynthesis in bread wheat (Triticum aestivum L.).

Resistant starch (RS) also known as healthy starch has shown several health benefits. Enhancing the RS through genetic modification approaches has huge commercial importance. Regulatory RNA like long non-coding RNA (lncRNA) plays an important role in gene regulation. In this study, we mined 63 transcriptome datasets of wheat belonging to 35 genotypes representing two seed developmental stages. Contrasting expression of a subset of lncRNAs in RS mutant lines compared to parent wheat variety 'C 306' signifies their probable role in RS biosynthesis. Further, lncRNA- TCONS_00130663 showed strong positive correlation (r2 = 1) with LYPL gene and strong negative correlation with SBEIIb (r2 = -0.94). We found TCONS_00130663 as positive regulator of LYPL gene through interaction with miR1128. Based on relative expression, in silico interaction and DSC analysis we hypothesize the dual role of TCONS_00130663 in RS type 2 and type 5. The study provides a useful resource for functional mechanism of lncRNAs.

Plant Transformation Techniques: Agrobacterium- and Microparticle-Mediated Gene Transfer in Cereal Plants.

Biotechnological methods for targeted gene transfers into plants are key for successful breeding in the twenty-first century and thus essential for the survival of humanity. Two decades ago, genetic transformation of crop plants was not routine, and it was all but impossible with important cereals such as barley and wheat. The recent focus on crop plant genomics-yet based on the Arabidopsis toolbox-boosted the research for more efficient plant transformation protocols, thereby considerably widened the number of genetically tractable crops. Moreover, modern genome editing methods such as the CRISPR/Cas technique are game changers in plant breeding, though heavily dependent on technical optimization of plant transformation. Basically, there are two successful ways of introducing DNA into plant cells: one is making use of a living DNA vector, namely, microbes such as the soil bacterium Agrobacterium tumefaciens that infects plants and naturally transfers and subsequently integrates DNA into the plant genome. The other method uses a direct physical transfer of DNA by means of microinjection, microprojectile bombardment, or polymers such as polyethylene glycol. Both ways subsequently require sophisticated strategies for selecting and multiplying the transformed cells under tissue culture conditions to develop into a fully functional plant with the new desirable characteristics. Here we discuss practical and theoretical aspects of cereal crop plant transformation by Agrobacterium-mediated transformation and microparticle bombardment. Using immature embryos as explants, the efficiency of cereal transformation is compelling, reaching today up to 80% transformation efficiency.

Biolistic Delivery of Programmable Nuclease (CRISPR/Cas9) in Bread Wheat.

The discovery of site-specific programmable nucleases has led to a major breakthrough in the area of genome editing. In the past few years, CRISPR/Cas system has been utilized for genome editing of a large number of crops including cereals like wheat, rice, maize, and barley. In terms of consumption, wheat is second only to rice as the most important crop of the world. In the present chapter, we describe biolistic delivery method of ribonucleoprotein (RNP) complexes of programmable nuclease (CRISPR/Cas9) for targeted genome editing and selection-free screening of transformants in wheat. The method not only overcomes the problem of random integration into the genome but also reduces the off-targets. Besides the step-by-step protocol, plausible challenges and ways to overcome them are also discussed. By using the described method of biolistic delivery of CRISPR/Cas9 in plant systems, genome-edited plants can be identified within 11 weeks.

Impact of polystyrene nanoplastics (PSNPs) on seed germination and seedling growth of wheat (Triticum aestivum L.).

Microplastics and nanoplastics are emerging pollutants of global concern. However, the understanding of their ecological effects on terrestrial plants is still limited. We conducted the systematic research to reveal the impact of polystyrene nanoplastics (PSNPs) (0.01-10 mg/L) on seed germination and seedling growth of wheat (Triticum aestivum L.). The results showed that PSNPs had no discernible effect on seed germination rate whereas significantly (p < 0.01) increased root elongation by 88.6 %-122.6 % when compared with the control. Similarly, remarkable increases in carbon, nitrogen contents, and plant biomass were also observed after exposure to PSNPs. Moreover, PSNPs could reduce the shoot to root biomass ratio (S:R ratio) of wheat seedlings. Furthermore, the imagings of a 3D laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM) indicated that PSNPs were taken up and subsequently down-top transported to shoot. The absorption and accumulation of four micronutrients (Fe, Mn, Cu and Zn) in wheat were generally reduced in varying degrees. Notably, metabolomics analysis revealed that all PSNPs treatments altered the leaf metabolic profiles mainly by regulating energy metabolisms and amino acid metabolisms. These findings are expected to provide new insights into the effects of PSNPs on crop plants.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

The functional analysis of a wheat group 3 late embryogenesis abundant protein in Escherichia coli and Arabidopsis under abiotic stresses.

Late embryogenesis abundant (LEA) proteins are highly hydrophilic and thermostable proteins that could be induced by abiotic stresses in plants. Previously, we have isolated a group 3 LEA gene WZY3-1 (GenBank: KX090360.1) in wheat. In this study, the recombinant plasmid with WZY3-1 was transformed into Escherichia coli BL21 for protein expression. Furthermore, we transformed WZY3-1 into Arabidopsis. Overexpression of WZY3-1 in E.coli enhanced their tolerance to mannitol and NaCl. WZY3-1 protein could protect lactate dehydrogenase (LDH) under freeze and heat stress. Overexpression of WZY3-1 showed that WZY3-1 could help to improve the drought tolerance of transgenic Arabidopsis. In summary, our works show that WZY3-1 plays an important role in abiotic stress resistance in prokaryotic and eukaryotic organisms.

Gradients of Gluten Proteins and Free Amino Acids along the Longitudinal Axis of the Developing Caryopsis of Bread Wheat.

Gradients in the contents and compositions of gluten proteins and free amino acids and the expression levels of gluten protein genes in developing wheat caryopses were determined by dividing the caryopsis into three longitudinal sections, namely, proximal (En1), middle (En2), and distal (En3) to embryo. The total gluten protein content was lower in En1 than in En2 and En3, with decreasing proportions of HMW-GS, LMW GS, and α/ß- and γ-gliadins and increasing proportions of ω-gliadins. These differences were associated with the abundances of gluten protein transcripts. Gradients in the proportions of the gluten protein polymers which affect dough processing quality also occurred, but not in total free amino acids. Microscopy showed that the lower gluten protein content in En1 may have resulted, at least in part, from the presence of modified cells in the dorsal part of En1, but the reasons for the differences in composition are not known.

Effects of ten years organic and conventional farming on early seedling traits of evolving winter wheat composite cross populations.

Early vigour traits of wheat composite cross populations (CCPs) based on high yielding (Y) or high quality (Q) or Y*Q varietal intercross evolving under organic or conventional conditions in parallel populations were studied hydroponically. To eliminate storage and year effects, frozen F6, F10, F11 and F15 seeds were multiplied in one field, resulting in the respective Fx.1 generations. This eliminated generation and growing system effects on seed size for the F6.1 F10.1 and F15.1. Due to a severe winter kill affecting the F11, the generation effect persisted, leading to larger seeds and markedly different seedling traits in the F11.1 compared to the F10.1 and F15.1. Seedling traits were similar among parallel populations. Shoot length and weight increased in both systems until the F11.1 across farming systems and remained constant thereafter. Over time, seminal root length and root weight of organic CCPs increased and total- and specific- root length decreased significantly compared to the conventional CCPs. Rooting patterns under organic conditions suggests better ability to reach deeper soil nutrients. In both systems, Q and YQ CCPs were more vigorous than Y CCPs, confirming genetic differences among populations. Overall, heterogeneous populations appear very plastic and selection pressure was stronger in organic systems.

Cell Wall Proteome of Wheat Grain Endosperm and Outer Layers at Two Key Stages of Early Development.

The cell wall is an important compartment in grain cells that fulfills both structural and functional roles. It has a dynamic structure that is constantly modified during development and in response to biotic and abiotic stresses. Non-structural cell wall proteins (CWPs) are key players in the remodeling of the cell wall during events that punctuate the plant life. Here, a subcellular and quantitative proteomic approach was carried out to identify CWPs possibly involved in changes in cell wall metabolism at two key stages of wheat grain development: the end of the cellularization step and the beginning of storage accumulation. Endosperm and outer layers of wheat grain were analyzed separately as they have different origins (maternal and seed) and functions in grains. Altogether, 734 proteins with predicted signal peptides were identified (CWPs). Functional annotation of CWPs pointed out a large number of proteins potentially involved in cell wall polysaccharide remodeling. In the grain outer layers, numerous proteins involved in cutin formation or lignin polymerization were found, while an unexpected abundance of proteins annotated as plant invertase/pectin methyl esterase inhibitors were identified in the endosperm. In addition, numerous CWPs were accumulating in the endosperm at the grain filling stage, thus revealing strong metabolic activities in the cell wall during endosperm cell differentiation, while protein accumulation was more intense at the earlier stage of development in outer layers. Altogether, our work gives important information on cell wall metabolism during early grain development in both parts of the grain, namely the endosperm and outer layers. The wheat cell wall proteome is the largest cell wall proteome of a monocot species found so far.

Label-free quantitative proteomic analysis of drought stress-responsive late embryogenesis abundant proteins in the seedling leaves of two wheat (Triticum aestivum L.) genotypes.

Late embryogenesis abundant (LEA) proteins are highly hydrophilic proteins with key roles in environmental stress responses. In this study, we performed the first survey of the LEA proteome in seedling leaves from two wheat genotypes subjected to drought stress, i.e., Shaanhe 6 (SH, drought-tolerant) and Zhengyin 1 (ZY, drought-sensitive). After isolating the LEA subpopulation by treating total soluble proteins with heating combined with 1% trichloroacetic acid treatment that was assessed by Western blotting of dehydrins, label-free proteomic analysis identified 38 LEA proteins or homologues belonging to seven LEA subfamilies in the two genotypes. The abundances of over half of the LEA proteins changed significantly after drought stress and they were involved in protection against drought, with at least 20 in SH and 14 in ZY. We found that the common differentially expressed LEA proteins increased in abundance more in the SH genotype compared with the ZY genotype, and six LEA proteins were significantly upregulated exclusively in the SH genotype, which may contribute to higher drought tolerance in SH. We also identified 221 non-LEA proteins from 12 functional categories. Our results provide a deeper understanding of the LEA expression patterns in response to drought stress in two wheat genotypes. SIGNIFICANCE: We identified 38 LEA proteins or homologues from different LEA families in two wheat genotypes, thereby indicating the complex and versatile protective roles of LEA proteins in drought stress resistance. Moreover, the abundance of differentially expressed LEA proteins increased more in the SH genotype compared with the ZY genotype, and several LEA proteins with significant upregulation only in the SH genotype may contribute to its higher tolerance of drought stress. 221 non-LEA proteins were differentially accumulated in at least one of the SH and ZY genotypes. They are involved mainly with 12 biological functions and they might explain different drought responses of the two genotypes. The differentially expressed LEA and non-LEA proteins may be potential markers of drought tolerance to facilitate wheat breeding, particularly those that were specifically upregulated in the SH genotype, or with opposing expression patterns in the two genotypes.

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.