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Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.

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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.

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The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.

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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.

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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.

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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.

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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.

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Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.

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Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

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Somatic embryogenesis in Agave genus has been induced; however, it is desirable to increase the rate of growth to get a more efficient propagation system. In this chapter, we present in detailed a protocol for somatic embryogenesis in Agave cupreata and the use of silver nanoparticles in a temporary immersion system. This is an efficient method that can be used commercially to improve the production and germination of somatic embryos.

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For the purpose of understanding the molecular processes triggered during callus formation in macaw palm, the expression of seven genes potentially involved in this process, identified in previous studies and from the literature, was investigated by RT-qPCR. In addition, in situ hybridization of the SERK gene was performed. Leaf tissues from adult plants from two macaw palm accession were inoculated in a medium combined with Picloram at a concentration of 450 µM to induce callus. The expression analysis was performed from leaf samples from two accessions of different origins (Municipalities of Tiros, MG, and Buriti Vermelho, DF, Brazil), which are characterized as non-responsive (NR) and responsive (R), respectively. The material was collected before callus induction (0 DAI, initial day) and 120 days after callus induction (120 DAI). Genes related to development (SERK, OASA, EF1, ANN1) and stress (LEA, CAT2, and MDAR5) were evaluated. The results obtained showed that all the genes involved with the development had their expressions downregulated at 0 DAI when the accession R was compared with the accession NR. On the other hand, it was possible to observe that these genes were upregulated at 120 DAI. The LEA stress gene showed a tendency to increase expression in the NR accession, while the R accession showed decreased expression and the CAT2 and MDAR5 genes showed upregulation in both accessions. In situ hybridization showed SERK transcripts in the vascular bundles, indicating the expression of SERK in this region, in addition to its expression in calluses. The results obtained in this study support our hypothesis that the regulation of genes involved in the control of oxidative stress and development is crucial for the formation of calluses in macaw palm.

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Plant growth regulators (PGR) are essential for somatic embryogenesis (SE) in different species, and Coffea canephora is no exception. In our study model, previously, we have been able to elucidate the participation of various genes involved in SE by using different strategies; however, until now, we have not used a proteomic approach. This research seeks to contribute to understanding the primary cellular pathways involved in developing SE in C. canephora. The process of our model consists of two stages: (1) preconditioning in MS medium with auxin (NAA) and cytokinin (KIN), and (2) induction in Yasuda liquid medium added with cytokinin (BA). Therefore, in this study, we analyzed different days of the SE induction process using shotgun label-free proteomics. An amount of 1630 proteins was found among different sampling days of the process, of which the majority were accumulated during the induction stage. We found that some of the most enriched pathways during this process were the biosynthesis of amino acids and secondary metabolites. Eighteen proteins were found related to auxin homeostasis and two to cytokinin metabolism, such as ABC, BIG, ILR, LOG, and ARR. Ten proteins and transcription factors related to SE were also identified, like SERK1, SKP1, nuclear transcription factor Y, MADS-box, and calreticulin, and 19 related to other processes of plant development, among which the 14-3-3 and PP2A proteins stand out. This is the first report on the proteomic approach to elucidate the mechanisms that operate during the induction of SE in C. canephora. So, our findings provide the groundwork for future, more in-depth research. Data are available via ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD047172.

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BACKGROUND: Rubber plant (Hevea brasiliensis) is one of the major sources of latex. Somatic embryogenesis (SE) is a promising alterative to its propagation by grafting and seed. Phytohormones have been shown to influence SE in different plant species. However, limited knowledge is available on the role of phytohormones in SE in Hevea. The anther cultures of two Hevea genotypes (Yunyan 73477-YT and Reken 628-RT) with contrasting SE rate were established and four stages i.e., anthers (h), anther induced callus (y), callus differentiation state (f), and somatic embryos (p) were studied. UPLC-ESI-MS/MS and transcriptome analyses were used to study phytohormone accumulation and related expression changes in biosynthesis and signaling genes. RESULTS: YT showed higher callus induction rate than RT. Of the two genotypes, only YT exhibited successful SE. Auxins, cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), gibberellins (GAs), and ethylene (ETH) were detected in the two genotypes. Indole-3-acetic acid (IAA), CKs, ABA, and ETH had notable differences in the studied stages of the two genotypes. The differentially expressed genes identified in treatment comparisons were majorly enriched in MAPK and phytohormone signaling, biosynthesis of secondary metabolites, and metabolic pathways. The expression changes in IAA, CK, ABA, and ETH biosynthesis and signaling genes confirmed the differential accumulation of respective phytohormones in the two genotypes. CONCLUSION: These results suggest potential roles of phytohormones in SE in Hevea.

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Somatic embryogenesis (SE) is an excellent example of mass plant propagation. Due to its genetic variability and low somaclonal variation, coffee SE has become a model for in vitro propagation of woody species, as well as for large-scale production of vigorous plants that are advantageous to modern agriculture. The success of the large-scale propagation of an embryogenic system is dependent on the development, optimization, and transfer of complementary system technologies. In this study, two successful SE systems were combined with a SETIS™ bioreactor immersion system to develop an efficient and cost-effective approach for the in vitro development of somatic embryos of Coffea spp. This study used an efficient protocol for obtaining somatic embryos, utilizing direct and indirect SE for both C. canephora and C. arabica. Embryos in the cotyledonary stage were deposited in a bioreactor to complete their stage of development from embryo to plant with minimal manipulation. Following ten weeks of cultivation in the bioreactor, complete and vigorous plants were obtained. Different parameters such as fresh weight, length, number of leaves, and root length, as well as stomatal index and relative water content, were recorded. In addition, the survival rate and ex vitro development of plantlets during acclimatization was assessed. The best substrate combination was garden soil (GS), peat moss (PM), and agrolite (A) in a 1:1:0.5 ratio, in which the bioreactor-regenerated plants showed an acclimatization rate greater than 90%. This is the first report on the use of SETIS™ bioreactors for the in vitro development of somatic embryos in Coffea spp., providing a technology that could be utilized for the commercial in vitro propagation of coffee plants. A link between research and innovation is necessary to establish means of communication that facilitate technology transfer. This protocol can serve as a basis for the generation and scaling of different species of agroeconomic importance. However, other bottlenecks in the production chains and the field must be addressed.

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This study aimed to establish a cryopreservation protocol for G. chacoensis embryogenic cultures (ECs) and to investigate the role of antioxidant enzymes activities during cryopreservation. The growth dynamics of cell suspensions were also investigated, followed by a phytotoxicity test to assess the ECs' ability to tolerate the use of cryoprotective solutions for different incubation times (0, 30, 60, 120, and 240 min). We evaluated the EC redox state in three steps of cryopreservation: after incubation in cryoprotection solution, after thawing, and 60 days after regrowth. Our results showed that the ECs support the use of cryoprotective solution until 120 min, showing phytotoxic effects with 240 min of incubation. This study reports a 100% survival of the cultures and a 10% increase ratio in fresh material for both incubation times tested (60 and 120 min). Increased malonaldehyde content was identified after incubation in the cryoprotective solution. An increase in the activities of catalase and ascorbate peroxidase was also identified in the subsequent steps, suggesting that the activation of antioxidant enzymes is essential for maintaining cell homeostasis during cryopreservation.

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Somatic embryogenesis (SE) involves modifications of cellular, biochemical, genetic, and epigenetic patterns. Our work investigated proteins as markers of embryogenic response and characterized the redox state of embryogenic cultures (EC) of Guadua chacoensis. We identified a total of 855 proteins; 129 were up- and 136 down-accumulated in EC as compared with non-embryogenic culture (NEC). Additionally, 37 and 22 proteins were identified as unique in EC and NEC, respectively. Heat-shock proteins as unique proteins and increased activity in Superoxide Dismutase and Guaiacol Peroxidase in EC suggest that the embryogenic response requires activation of the stress response mechanism. Ribosomal, translational, and glycolytic proteins in EC seem to be associated with protein synthesis and energy sources for embryo development, respectively. Accumulation of cell wall-related proteins, such as Arabinogalactan and Polygalacturonase inhibitors, and signaling transduction proteins, including Chitinase, Phospholipase, and Guanine nucleotide-binding proteins in EC seems to be associated with embryogenic response. Enhancement of H2O2 content in EC compared to NEC suggests a possible role as a secondary messenger in SE. Altogether, the present study identified marker proteins of embryogenic response in G. chacoensis and revealed the activation of ROS scavenging enzymes to assure cell redox homeostasis and SE responses. SIGNIFICANCE: Somatic embryogenesis is a promising technique for the propagation and conservation of bamboo species; however, this route has been the least understood and studied until now. This study corresponds to the first work approaching proteomics complemented with biochemical analyses in the somatic embryogenesis of bamboo, bringing robust and precise information that can improve our understanding of this complex morphogenetic route.

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Since the discovery of somatic embryogenesis (SE), it has been evident that nitrogen (N) metabolism is essential during morphogenesis and cell differentiation. Usually, N is supplied to cultures in vitro in three forms, ammonium (NH4+), nitrate (NO3-), and amino N from amino acids (AAs). Although most plants prefer NO3- to NH4+, NH4+ is the primary form route to be assimilated. The balance of NO3- and NH4+ determines if the morphological differentiation process will produce embryos. That the N reduction of NO3- is needed for both embryo initiation and maturation is well-established in several models, such as carrot, tobacco, and rose. It is clear that N is indispensable for SE, but the mechanism that triggers the signal for embryo formation remains unknown. Here, we discuss recent studies that suggest an optimal endogenous concentration of auxin and cytokinin is closely related to N supply to plant tissue. From a molecular and biochemical perspective, we explain N's role in embryo formation, hypothesizing possible mechanisms that allow cellular differentiation by changing the nitrogen source.

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In this work, we report a successful protocol to obtain in vitro peach palm (Bactris gasipaes Kunth) "Diamantes 10" plants through somatic embryogenesis from transverse thin cell layer (TCL) explants, dissected from three sections (basal, medial, and apical) of lateral offshoots of adult plants cultured on different concentrations of 4-amino-3,5,6-trichloropicolonic acid (picloram). After swelling and development of primary callus in all treatments, without any strong effect of explant origin or picloram concentration, it was possible to observe the formation of embryogenic structures and the exact point from where they developed. Browning was also observed and correlated to the induction treatments, although it was not an impairment for the production of embryogenic structures. Subsequent maturation and conversion of somatic embryos into plantlets allowed their acclimatization 17 months after culture initiation (ACI), which was quicker than previous reports with juvenile tissues (from embryos or seed-germinated plantlets). To the best of our knowledge, this is the first report on peach palm regeneration through somatic embryogenesis from TCL explants from adult plants and could constitute, after fine-tuning the acclimatization stage, a tool for mass clonal propagation of elite genotypes of this open-pollinated crop, as well as for the establishment of conservation strategies of in situ gene bank plant accessions endangered due to aging and other threats.

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Culture in temporary immersion systems (TIS) is a valuable tool for the semi-automation of high frequency somatic embryogenesis of coffee. This system allows the intermittent exposure of explants to liquid medium in cycles of specific frequency and duration of immersion with renewal of the culture atmosphere in each cycle. TIS have revolutionized somatic embryogenesis of coffee plants as an alternative for scaling up and reducing costs associated with labor-intensive solid media culture. In Central America, somatic embryogenesis is employed on a commercial scale to produce F1 Coffea arabica hybrids. In Asia and Africa, somatic embryogenesis is used for the multiplication of selected genotypes of C. arabica and C.canephora. Somatic embryogenesis of coffee plants is considered a model system for woody species due to its biological versatility and low frequency of somaclonal variation. Nevertheless, the success of somatic embryogenesis for mass propagation of coffee plants depends on the development, optimization, and transfer of complementary technologies. Temporary immersion using the RITA® bioreactor is, so far, the best complementary tool for somatic embryogenesis of Arabica coffee for a single recipient with simple changes in liquid media. Likewise, high volume bioreactors, such as 10-L glass BIT® and 10-L flexible disposable plastic bags, have been successfully used for somatic embryogenesis of other coffee species. These bioreactors allow the manipulation of thousands of embryos under semi-automated conditions. The protocols, advantages, and benefits of this technology have been well documented for organogenesis and somatic embryogenesis pathways. However, adaptation in commercial laboratories requires technical and logistical adjustments based on the biological response of the cultures as well as the costs of implementation and production. This review presents the historical and present background of TIS and its commercial application and, in particular, pertinent information regarding temporary immersion culture for C. arabica somatic embryogenesis. The main limitations of this technology, such as hyperhydricity, asynchrony, and developmental abnormalities, are examined, and a critical analysis of current knowledge regarding physiological, biochemical, and molecular aspects of the plant response to temporary immersion is offered. Further, perspectives are provided for understanding and solving the morpho-physiological problems associated with temporary immersion culture of coffee plants. Systematic Review Registration.

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Somatic embryogenesis is the process by which embryos are formed from a single or small group of somatic cells in response to specific stimuli. Somatic embryogenesis has been applied to achieve mass clonal propagation on an industrial scale and to increase the agronomic performance of species of economic interest, including sugarcane. The use of somatic embryogenesis in sugarcane stands out as a biotechnological tool with a high potential for application in the clonal propagation of disease-free elite varieties, as an essential part of genetic transformation protocols, and in the production of synthetic seeds. A better understanding of each phase of somatic embryogenesis can help to optimize the process to enhance yields and produce high-quality emblings. In this chapter, we describe a detailed protocol for somatic embryogenesis in sugarcane (Saccharum sp.) to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment of sugarcane emblings.

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