Plant Micropropagation and Temporary Immersion Systems.

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.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Passiflora spp. (Passifloraceae).

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.

Protocol for the Acclimatization of Coconut Vitro-Plants Under Ex Vitro Conditions.

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.

Scale-Up of Coffea canephora Somatic Embryogenesis in Temporary Immersion System.

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.

Advances in Tissue Culture and Transformation Studies in Non-model Species: Bixa orellana L. (Bixaceae).

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.

Direct Somatic Embryogenesis in Carica papaya L. Genotypes for Genetic Modification Purposes.

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.

Transcriptomic Analysis During the Induction of Somatic Embryogenesis in Coffea canephora.

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.

An Introduction to Plant Cell, Tissue, and Organ Culture: Current Status and Perspectives.

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.

Lipids in Insect Reproduction: Where, How, and Why.

Modern insects have inhabited the earth for hundreds of millions of years, and part of their successful adaptation lies in their many reproductive strategies. Insect reproduction is linked to a high metabolic rate that provides viable eggs in a relatively short time. In this context, an accurate interplay between the endocrine system and the nutrients synthetized and metabolized is essential to produce healthy offspring. Lipids guarantee the metabolic energy needed for egg formation and represent the main energy source consumed during embryogenesis. Lipids availability is tightly regulated by a complex network of endocrine signals primarily controlled by the central nervous system (CNS) and associated endocrine glands, the corpora allata (CA) and corpora cardiaca (CC). This endocrine axis provides hormones and neuropeptides that significatively affect tissues closely involved in successful reproduction: the fat body, which is the metabolic center supplying the lipid resources and energy demanded in egg formation, and the ovaries, where the developing oocytes recruit lipids that will be used for optimal embryogenesis. The post-genomic era and the availability of modern experimental approaches have advanced our understanding of many processes involved in lipid homeostasis; therefore, it is crucial to integrate the findings of recent years into the knowledge already acquired in the last decades. The present chapter is devoted to reviewing major recent contributions made in elucidating the impact of the CNS/CA/CC-fat body-ovary axis on lipid metabolism in the context of insect reproduction, highlighting areas of fruitful research.

In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

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.

Prenatal post-implantation development of collared peccaries (Pecari tajacu Linnaeus, 1758).

Given the importance of information on intrauterine development in diagnosing anomalies in the gestational development of the species for the development of assisted reproduction technologies as well as understanding the autonomy and responsiveness of the newborn, the aim of the present study was to describe the external morphology of collared peccary conceptuses. For this study, two conceptuses were used per gestational age of 25-120 days post-copulation (dpc) and neonates with 145 dpc, totalling 22 animals. Females were euthanised, and embryos/foetuses were examined, measured, and photographed. During the first third of the gestational period (25-50 dpc, n = 8), a marked body curvature, brain vesicles, somites, internal organs, placid lens, auricular protrusion and limb buds are noted. In the second third of the gestational period (51-100 dpc, n = 10), foetuses lose their body curvature, displaying greater anatomical definition, including skeletal, external ears, nostrils, eyelids and tactile hair formation and cranial suture closure. In addition, dorsal scent gland and genital tubercle differentiation were visualized at 50 days post-copulation. In the third of the gestational period (101-145 dpc, n = 4), the organs become completely formed, alongside skin darkening, eyelid opening, dental eruption, dorsal odorous gland development, sexual organ externalization, and fanero attachment development. These data allowed for the construction of a prenatal growth curve, providing comparative anatomy information for ungulates and further contributing towards rational reproductive management and reproductive biotechnologies for this species.

In-nest mortality and pathology of hawksbill sea turtle (Eretmochelys imbricata) embryos and hatchlings in St. Kitts and Nevis.

Strategies to improve the hatch success and survival of critically endangered hawksbill turtles (Eretmochelys imbricata) require knowledge of threats to them including pathological conditions. The objective of this study was to describe the mortality and pathology of embryos and dead-in-nest hatchlings on St. Kitts and Nevis. Over the 2019/20 and 2020/21 nesting seasons, the combined mean (SD) hatch success for the two islands was 81.9% (13.2%) and deceased individuals within excavated nests were early-stage embryos (70.7%), late-stage embryos (17.7%), pipped-hatchlings (8.2%) or dead-in-nest hatchlings (3.4%). From 2017 to 2021, a post-mortem examination was performed on 183 turtles, including histology for 116. Anatomical malformations affected 77 (42.1%) examined turtles and included abnormal scute shape or number (22.4%), dysmelia (8.7%), schistosomus reflexus (7.7%) and compressed carapace (7.7%). Microscopic lesions were found in 49.1% of turtles and included tissue mineralization (26.7%, including renal, fetal membrane, liver, heart or muscle), chorioallantoitis (16.2%) and skeletal muscle degeneration and necrosis (10%). Inflammatory lesions associated with fungal or bacterial infections were in the skin (n = 3), chorioallantois (n = 4), lung (n = 3) or yolk sac (n = 1). These lesions may reflect non-specific terminal conditions but their presence in-nest helps explain some of the mortality and pathology documented in hatchlings that die during rehabilitative care. All of the gonads adequately represented for histological determination of sex were female (n = 62), supporting concern for feminization of Caribbean hawksbill turtle nests. The study identifies lesions that could affect hatch and emergence success. The high frequency of skeletal malformations indicates the need for investigations addressing regional impact and pathogenesis, especially genetic and environmental aetiologies including nest temperature. Immediate examination of live hatchlings on nest emergence is warranted to better determine the prevalence of non-fatal malformations that could impact fitness and population genetics.

Plant Regeneration of Agave cupreata by Somatic Embryogenesis in a Temporary Immersion System with Silver Nanoparticles.

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.

Secondary Embryogenesis of Linaloe in Temporary Immersion Bioreactor-Type RITA®.

The linaloe [Bursera linanoe (La Llave) Rzed, Calderon and Medina] is an endemic species of Mexico, representative of the low deciduous forest of the states of Guerrero, Puebla, Morelos, and Oaxaca, and has been of great economic importance for the people, mainly for the artisanal use of its aromatic wood that is used to make boxes, trunks, and furniture that are manufactured in Olinala, Guerrero, Mexico; and industrial, thanks to the fine aroma of its essential oil (linalool), which is used in the manufacture of perfumes and pharmaceuticals. Overexploitation has endangered the species in recent years, and propagation by seed and/or cuttings has produced very poor results compared to those obtained with other recalcitrant Bursera species. The protection of endangered species makes urgent the need to propose new alternatives for its propagation. Somatic embryogenesis is a reliable and feasible technique, including induction, maintenance, multiplication, and maturation of embryos, often in semisolid culture media; however, the recent use of liquid media has allowed semi-automation in temporary immersion bioreactors, for example, the RITA® system, which favors both the multiplication rate and the final conversion to seedlings.

Differential expression of genes potentially related to the callogenesis and in situ hybridization of SERK gene in macaw palm (Acrocomia aculeata Jacq.) Lodd. ex Mart.

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.

Proteomic Approach during the Induction of Somatic Embryogenesis in Coffea canephora.

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.

Comparative transcriptome analysis of high- and low-embryogenic Hevea brasiliensis genotypes reveals involvement of phytohormones in somatic embryogenesis.

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.

Essential Oils Produce Developmental Toxicity in Zebrafish Embryos and Cause Behavior Changes in Zebrafish Larvae.

Essential oils have gained significant popularity in various industries due to their biological properties, but their potential toxic effects on living organisms have been poorly investigated. This study aimed to evaluate the effects of lemongrass, thyme, and oregano essential oils on zebrafish embryos and larvae as animal models. Embryos were exposed to different concentrations of essential oils, and various endpoints were assessed, including epiboly, mortality (LC50), morphometry, and behavioral changes. All three essential oils reduced epiboly, affecting embryonic development. LC50 values were calculated for lemongrass (3.7 µg/mL), thyme (14.4 µg/mL), and oregano (5.3 µg/mL) oils. Larvae exposed to these oils displayed morphological defects, including growth reduction, spinal deformation, pericardial edema, eye size reduction, and reduced swim-bladder inflation. Morphometric analysis confirmed reduced larval length at higher oil concentrations. Essential-oil exposure altered zebrafish larval swimming behavior, with lemongrass oil reducing dark-cycle activity and oregano oil increasing light-cycle activity, suggesting neurodevelopmental toxicity. These findings illustrate the adverse effects of these oils on zebrafish embryos and larvae and reveal essential-oil toxicity, indicating careful use should be considered, particularly during pregnancy.

In Vitro Conversion of Coffea spp. Somatic Embryos in SETIS™ Bioreactor System.

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.

Effect of the Aqueous Extract of Chrysobalanus icaco Leaves on Maternal Reproductive Outcomes and Fetal Development in Wistar Rats.

Toxicological studies on medicinal plants are essential to ensure their safety and effectiveness in treating various diseases. Despite the species Chrysobalanus icaco L. being popularly used in the treatment of several diseases due to the pharmacological properties of its bioactive compounds, there are few studies in the literature regarding its toxicity regarding reproduction. Therefore, the purpose of this study was to assess the potential embryotoxic and teratogenic effects of the aqueous extract of C. icaco leaves (AECi) on Wistar rats. Animals were given AECi at doses of 100, 200, and 400 mg/kg during the pre-implantation and organogenesis periods. Data were analyzed using ANOVA followed by Tukey's test and Kruskal-Wallis. Pregnant rats treated during the pre-implantation period showed no signs of reproductive toxicity. Rats that received AECi at 100, 200, and 400 mg/kg during organogenesis did not exhibit any signs of maternal systemic toxicity or significant differences in gestational and embryotoxic parameters. Some skeletal changes were observed in the treated groups. Therefore, it can be suggested that AECi at doses of 100, 200, and 400 mg/kg is safe for treated animals and does not induce reproductive toxicity under the experimental conditions applied, but it also caused low systemic toxicity.