Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency.

BACKGROUND: Somatic embryogenesis (SE) exemplifies the unique developmental plasticity of plant cells. The regulatory processes, including epigenetic modifications controlling embryogenic reprogramming of cell transcriptome, have just started to be revealed.
RESULTS: To identify the genes of histone acetylation-regulated expression in SE, we analyzed global transcriptomes of Arabidopsis explants undergoing embryogenic induction in response to treatment with histone deacetylase inhibitor, trichostatin A (TSA). The TSA-induced and auxin (2,4-dichlorophenoxyacetic acid; 2,4-D)-induced transcriptomes were compared. RNA-seq results revealed the similarities of the TSA- and auxin-induced transcriptomic responses that involve extensive deregulation, mostly repression, of the majority of genes. Within the differentially expressed genes (DEGs), we identified the master regulators (transcription factors - TFs) of SE, genes involved in biosynthesis, signaling, and polar transport of auxin and NITRILASE-encoding genes of the function in indole-3-acetic acid (IAA) biosynthesis. TSA-upregulated TF genes of essential functions in auxin-induced SE, included LEC1/LEC2, FUS3, AGL15, MYB118, PHB, PHV, PLTs, and WUS/WOXs. The TSA-induced transcriptome revealed also extensive upregulation of stress-related genes, including those related to stress hormone biosynthesis. In line with transcriptomic data, TSA-induced explants accumulated salicylic acid (SA) and abscisic acid (ABA), suggesting the role of histone acetylation (Hac) in regulating stress hormone-related responses during SE induction. Since mostly the adaxial side of cotyledon explant contributes to SE induction, we also identified organ polarity-related genes responding to TSA treatment, including AIL7/PLT7, RGE1, LBD18, 40, HB32, CBF1, and ULT2. Analysis of the relevant mutants supported the role of polarity-related genes in SE induction.
CONCLUSIONS: The study results provide a step forward in deciphering the epigenetic network controlling embryogenic transition in somatic cells of plants.

This study leads with the primed seeds of rice (var. Swarna) with distilled water (D.W.) and various concentrations of Mg(NO3)2 (0-8 mM)/Kinetin (0-5 ppm) alone or in combination with screen out the regeneration medium induced tolerance level of NaCl. To fulfill the objective, the primed and non-primed rice seeds were inoculated in MS medium supplemented with 30 gL-1 maltose + 1 gL-1 casein hydrolysate and 2 mgL-1 of 2,4-D for callus induction and cultured up to 45 days in two sets: one set for regeneration purpose in NaCl-induced regeneration medium and another set was used to study the physiological potentiality of the callus. The 45-day-old calli were transferred into regeneration medium MSR (MS medium for regeneration) (BAP: NAA: Kinetin = 4:1:1) containing NaCl with a concentration range of 0 to 300 mM. The number of regenerating calli and shoot regeneration percentage, number of plantlets obtained from one callus, recovery of plantlets from each concentration of NaCl and proline estimation from the leaf of the regenerated plantlets were determined from one set obtained after 45 days. The calli obtained from another set after 45 days, the frequencies of total and embryogenic calli induction percentage, fresh and dry weights, proline content, nitrate reductase and superoxide dismutase activities were measured. The calli obtained from 2.5 ppm kinetin + 4 mM Mg(NO3)2 primed seeds were showed best result as compared to the other treatments for the above-mentioned parameters in different concentrations of NaCl-induced medium and survive up to 200 mM concentrations of NaCl.

Quercus aliena, a native Chinese tree species, is significant in industry and landscaping. However, it is traditionally propagated by seeds with many limitations, such as pest infestations, seed yield and quality. Thus, this study firstly introduces a somatic embryogenesis (SE) system for Q. aliena, enhancing its cultivation prospects. Thereinto, the development stage of zygotic embryo had a significant effect on SE, only immature embryos in 10-11 weeks after full bloom (WAF), rich in endogenous abscisic acid (ABA), could induce SE. Exogenous application ABA had positive roles in the early development process of both primary and secondary SE, while its antagonist had opposite roles. Transcriptome analysis showed that transcription regulation occupied the major position. Mfuzz cluster and WGCNA co-expression analysis showed that 24 candidate genes were involved in the SE process. The expression of the 24 genes were also affected by exogenous ABA signals, among which QaLEC2, QaCALS11 and QaSSRP1 occupied the important roles. Additionally, the callose content were also affected by exogenous ABA signals, which had significantly positive correlations with the expression of QaLEC2 and QaCALS11. This study not only established an efficient reproduction system for Q. aliena, but also revealed the difference in embryogenic ability of zygotic embryos from the aspects of transcriptome and endogenous hormone content, and lay a foundation for clarifying the molecular mechanism of SE, and provided a reference for exploring the vital roles of ABA in SE.

Plant growth regulators (PGRs) play a vital role in the induction of morphogenesis in vitro. Synthetic PGRs are commonly used to induce organogenesis and somatic embryogenesis from various explants, while natural substances are rarely utilized. This study aimed to enhance the regenerative response in Nicotiana tabacum leaf explants using Tulsi (Ocimum sanctum) leaf extract and to elucidate the biochemical interactions during modulation of endogenous plant growth regulators, including indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin, and 6-(γ, γ-dimethylallylamino) purine (2iP). Tulsi leaf extract significantly improved shoot production through interactions between endogenous hormones and those present in the extract, which enhanced stress mitigation. The 20% Tulsi leaf extract treatment produced significantly more shoots than the control, coinciding with increased endogenous IAA and zeatin levels starting on day 10 in culture. Furthermore, ABA and zeatin concentrations increased on days 15 and 25, respectively, in the 20% Tulsi extract treatment, suggesting their role in the induction of somatic embryo-like structures. ABA likely acts as an activator of stress responses, encouraging the development of these structures. Additionally, 2iP was involved in the induction of both forms of regeneration in the 10% and 20% extract treatments, especially in combination with ABA. These results suggest that Tulsi leaf extract holds promising potential as a natural supplement for increasing plant regeneration in vitro and advancing our understanding of how natural extracts of plant origin can be harnessed to optimize plant regeneration processes in vitro.

Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.

Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species\' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.