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.

Effects of plant growth regulators and sucrose on proliferation and quality of embryogenic tissue in Picea pungens.

Embryogenic tissue (ET) is important for genetic modification and plant re-generation. The proliferation ability and vigor of ET are crucial for plant propagation via somatic embryogenesis. In this study, ET was induced from mature zygotic embryos in blue spruce (Picea pungens Engelm.). There were significant differences in ET induction between two provenances, i.e. 78.8 ± 12.5% and 62.50 ± 12.8% respectively. Effects of 2,4-Dichlorophenoxy acetic acid (2,4-D), 6-Benzyl amino-purine (6-BA) and/or sucrose on ET proliferation and somatic embryo (SE) maturation were further investigated with four cell lines. The highest ET proliferation rate reached 1473.7 ± 556.0% biweekly. Concentrations of 2,4-D or 6-BA applied at tissue proliferation stage impacted SE maturation among the cell lines, whereas sucrose showed less effects. The highest rate, 408 ± 230 mature SEs/g FW, was achieved in SE maturation cultures. This research demonstrated that the culture conditions, i.e. the specific concentrations of 2,4-D and BA, at ET proliferation stage affected not only ET growth, but also the quality of ET for SE maturation. This study revealed the necessity and benefit in developing both the general and the genotype-specific protocols for efficient production of mature SEs, or somatic plants in blue spruce.

Comparative proteomic analysis and antioxidant enzyme activity provide new insights into the embryogenic competence of Guadua chacoensis (Bambusoideae, Poaceae).

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.

Single-base resolution methylomes of somatic embryogenesis in Theobroma cacao L. reveal epigenome modifications associated with somatic embryo abnormalities.

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

Overview of Somatic Embryogenesis.

Somatic embryogenesis is a natural phenomenon through which somatic embryos are produced from somatic cells although. It is considered the most efficient morphogenic pathways for plant multiplication. One of the key features of somatic embryogenesis is the use of cellular totipotency, where dedifferentiation is induced to foster cell proliferation, followed by the induction of differentiation using plant growth regulators to produce new plants. There is a cell group with the potential to undergo the somatic embryogenesis pathway through adequate stimulation (plant growth regulators, incubation conditions, and supplementation of the culture medium). There are two somatic embryogenesis pathways in plants: direct and indirect embryogenesis. Direct somatic embryogenesis consists of the formation of embryos directly from isolated cells, without the formation of "callous" tissue. Indirect somatic embryogenesis is characterized by the formation of a callus as a stage that precedes the formation of somatic embryos. It should be stressed that not all plant cells have this morphogenic capacity; consequently, determining the type of factors that drive this type of response has been challenging. This book provides the reader with updated available information on the techniques, relevant protocols, and tools to perform somatic embryogenesis in different plant species for economic purposes.

Theobroma cacao: Somatic Embryogenesis.

A two-step process combining direct and indirect somatic embryogenesis, on solid and liquid medium, respectively is described for Theobroma cacao L. Staminodes and petals from unopened bud flowers are used to induce primary direct embryos. Then, these primary embryos are cut to produce embryogenic calli which will develop secondary embryos. This step of indirect SE allows us to produce large quantities of embryos and to do mass propagation using liquid culture medium. Despite a very strong clone dependency and high batch-to-batch variability, about 80% of T. cacao cultivars respond to somatic embryogenesis and can be propagated by this method.

Use of Thin Cell Layer Technique for Induction of Somatic Embryogenesis of Agave fourcroydes.

Agave fourcroydes (henequén) is a plant used for the extraction of hard fiber from its leaves. Due to its long-life cycle, it is very difficult to genetically improve. Somatic embryogenesis (SE) is a very useful micropropagation technique, that can be used for genetic improvement programs and increase the micropropagation of this species. SE is a morphogenic process by which somatic embryos are generated from somatic cells reprogramming. To initiate the regeneration program, the loss of cell-cell communication is suggested to be important. The Thin Cell Layer (TCL) technique allows for the isolation of specific cell or tissue layers, and in conjunction with strictly controlled growth conditions, may lead to the in vitro induction of specific morphogenic programs. Here, we describe a new protocol for the induction of somatic embryogenesis through TCL culture technique, from stem of elite clonal A. fourcroydes vitroplants previously generated through micropropagation of adventitious shoots.

Somatic Embryogenesis in Banana (Musa spp.).

Bananas (Musa ssp.) are among the world's most important crops. In terms of gross value of production, they are the fourth most important global food crop and have an important socioeconomic and ecological role. Somatic embryogenesis (SE) is a developmental process, in which somatic cells differentiate into embryos which eventually develop and regenerate into plants. SE is exploited to generate a large quantity of very high economic value, genetically identical and disease-free plantlets. In bananas, the use of shoot apexes of axillary buds to induce SE resulted an alternative for plant regeneration through embryogenic cell suspension (ECS). The protocol has been scaled up to commercial laboratories for tissue culture (biofactories) for production of planting materials. The genetic stability of regenerated plants and high yields obtained under field conditions demonstrate the feasibility of scaling up this biotechnological protocol and adapting it to commercial production of planting materials to mitigate a critical bottleneck in the value chain of this important crop.

Citrus Protoplast Isolation and Plant Regeneration Through Somatic Embryogenesis.

Protoplasts are an attractive explant source for biotechnological tools widely used on citrus genetic improvement, such as somatic hybridization and direct genetic transformation. These delicate and responsive materials are subjected to cell proliferation induction and differentiation of somatic embryos which further regenerate into entire plants. The isolation of viable protoplasts followed by regeneration of plants through somatic embryogenesis is an important methodology for breeding applications. The methods presented here can also be used as a reference for protoplast work in other species, followed by protocol optimization for different species/genotypes.

Somatic Embryogenesis in Pines.

Among the different in vitro culture techniques, somatic embryogenesis has been one of the most important developments for plant tissue culture; it has enabled mass propagation and the development of biotechnological tools to enhance the productivity and quality of plantation forestry. This propagation technique together with cryopreservation is the base of multivarietal forestry.The development of somatic embryogenesis in forest trees dates from 1985, and in the last years several studies have focused on the development and optimization of the conifer somatic embryogenesis process to make it more efficient in terms of both the quantity and the characteristics of the plants obtained. However, these advances are not sufficiently refined to be implemented commercially for many Pinus spp. due to the high cost of the process derived from hand labor. Nowadays, trying to add value to the plants produced to compensate the high costs of the process, different studies are being developed in order to obtain Pinus somatic plants with better adaptation to environmental stresses prompted by the current situation of climate change.In this chapter, a summary of the recent somatic embryogenesis systems developed to achieve Pinus spp. high quality plants is presented.

Somatic Embryogenesis of Anthurium andraeanum Linden., -A Tropical Florists' Plant.

The global floriculture market is expected to reach US$41.1 billion by 2027 at a CAGR of 5% over the analysis period 2020-2027; on the year 2020, the recorded market value in this trade was US$29.2 billion. The florists mainly use Anthurium andraeanum flowers in fashionable bouquets and floral arrangements because of their beautiful, attractive bright colored eye-catching spathe, candle-like spadix, prolonged vase life, etc. The cut flower industry always seeks elite cultivars and new hybrids of A. andraeanum, that in turn depend on the availability of large numbers of clonal planting propagules. In vitro somatic embryogenesis is an important technique for large-scale clonal propagation, development of transgenic plants, creation of new variety by somaclonal variation, mutagenesis on in vitro plants, and germplasm preservation for future use. Here, we describe the protocol of somatic embryogenesis of Anthurium andraeanum cv. Cancan, an important commercial cultivated variety. The protocol has been optimized by using 4 different types of culture media which are used during embryogenic callus induction, multiplication of callus, induction of somatic embryogenesis, and maturation plus conversion of embryos into plantlets. The protocol outlines the detailed methods from mother plant procurement to hardening of micro plants that is ready to transfer in the field and it can be used for large-scale commercial propagation.

Somatic Embryogenesis in Citrus (Citrus spp.), var. Valencia.

Somatic embryogenesis has been obtained in many citrus cultivars; however, the efficiency of the system is genotype dependent and culture synchronization is important to reach more efficient systems. In this chapter we present a detailed protocol of somatic embryogenesis induction from nucellar tissue and the use of an alternative method of callus sieving for culture synchronization and embryo production. This is a simple method which can also be evaluated for other species aiming at better culture efficiency and somatic embryo production.

Somatic Embryogenesis in Papaya (Carica papaya L.).

High mortality rates of in vitro plants during ex vitro acclimatization, due to low rooting, is one of the main problems of papaya tissue culture. This work was carried out with the objective to obtain 100% hermaphroditic in vitro plants of the papaya cultivar "Maradol Roja" by somatic embryogenesis, which have an adequate rooting system that allows them a higher survival percentage in the ex vitro acclimatization phase. In international scientific literature, there are several protocols; however, not all of them cover the different phases of somatic embryogenesis. This chapter describes a complete and optimized protocol from immature zygotic embryos in this cultivar. It also looks at the morpho-anatomical characterization of somatic embryos in the different stages of ontogenetic development, as well as high survival rates under ex vitro conditions of the plants obtained. It can be used for genetic improvement and propagation of this species.

Use of Thin Cell Layer (TCL) to Obtain Somatic Embryogenesis.

The thin cell layer (TCL) culture system was initially reported in relation to the model plant Nicotiana tabacum, giving rise to 47 years of continuous application and investigation on micropropagation and plant breeding of over 100 plant species or hybrids. The small sizes of the tissue sections (100 µm to 1-2 mm in thickness), its classification into transverse TCL (tTCL) or longitudinal TCL (lTCL) categories, and the interaction between the cultured cells and the culture medium are the main drivers of its efficacy in tens of plants for the induction of somatic embryogenesis, relative to the conventional in-vitro culture system. Furthermore, it promotes higher productivity and reduced time in the proliferation of cultures, which is key for the differentiation of cells and plant tissues. This chapter describes the main characteristics of the TCL sections, and the interaction between cells under in-vitro culture. In addition, it highlights the latest findings reporting the success of TCL in ornamental, herbaceous, woody, and recalcitrant plants. In most cases, studies on the use of TCL in combination with techniques such as bioreactors, histology, genetic transformation, and fidelity analysis, provide indisputable evidence that highlights the importance of this technique in plant biotechnology. Finally, the perspectives on TCL use are described, underlining the advantages and constraints of the technique for its continued use and future application.

Perspectives of Somatic Embryogenesis: Concluding Remarks.

One of the main objectives to achieve in plant tissue culture is the multiplication of the available plant material, taking full advantage of the regenerative capacities of plant cells. Somatic embryogenesis leverages cell totipotency to produce new explants from a cell, thus obtaining many propagules for scientific research, industrial, or exploitation purposes. Somatic embryogenesis (ES) characterizes by being one of the most efficient techniques in plant micropropagation. However, developing an efficient plant ES protocol requires several key factors to consider, as demonstrated throughout the chapters of this book. These chapters highlight the major drivers of the success of ES in different plant species: plant growth regulators, concentration of auxins and cytokines, water deficit, photoperiod, and type of culture medium; techniques such as the use of bioreactors and Thin Cell Layer (TCL); and the influence of stress on the formation of somatic embryos. Research has been conducted to address each phase of somatic embryogenesis, either individually or for all phases. The chapters of this book cover in detail the techniques used and provide guidance that will allow readers to successfully develop all the somatic embryogenesis phases in different cultures, from cell dedifferentiation to differentiation.

Cellular responses of oil palm genotypes during somatic embryogenesis involve participation of procambial cells, DNA demethylation, and auxin accumulation.

KEY MESSAGE: Cell markers of somatic embryogenesis initiation from leaf tissues in oil palm involve the participation of procambial cells, DNA demethylation, and auxin accumulation. Low callogenesis and genotype-dependent response have been mentioned in the development of somatic embryogenesis protocols of Elaeis oleifera × E. guineensis elite hybrids, which requires more detailed investigations of the process. Thus, the initial cellular responses of immature leaves of adult genotypes of this hybrid were investigated for the first time, emphasizing histological, epigenetic, and endogenous auxin changes. Leaf segments from two genotypes, one responsive to somatic embryogenesis (B351733) and another non-responsive (B352933), were inoculated in Murashige and Skoog medium with 450 µM of 4-amino-3, 5, 6-trichloropicolinic acid. For anatomical analysis, samples of both genotypes were collected at 0, 20, 90, and 105 days of cultivation. Samples of both genotypes were also taken at different cultivation periods to analyze DNA methylation status (% 5-mC-5 methylcytosine) via ELISA test. Immunolocalization assays were performed with anti-indole-3-acetic acid and anti-5-methyl-deoxycytosine antibodies from samples of hybrid B351733. We distinguished two groups of cells reactive to the induction of embryogenic callogenesis, parenchymatous sheath cells, and procambial cells; however, only the latter are directly involved with the formation of calluses. The data obtained indicate that the formation of calluses in hybrid B351733 is related to DNA hypomethylation, while the non-responsiveness of leaf explants in hybrid B352932 is related to DNA hypermethylation. The in situ immunolocalization enabled the identification of initial markers of the callogenic process, such as IAA accumulation and hypomethylation. Identifying these events brings the possibility of establishing strategies for efficient manipulation of somatic embryogenesis protocols in palm trees.

Changing Temperature Conditions during Somatic Embryo Maturation Result in Pinus pinaster Plants with Altered Response to Heat Stress.

Under the global warming scenario, obtaining plant material with improved tolerance to abiotic stresses is a challenge for afforestation programs. In this work, maritime pine (Pinus pinaster Aiton) plants were produced from somatic embryos matured at different temperatures (18, 23, or 28 °C, named after M18, M23, and M28, respectively) and after 2 years in the greenhouse a heat stress treatment (45 °C for 3 h/day for 10 days) was applied. Temperature variation during embryo development resulted in altered phenotypes (leaf histology, proline content, photosynthetic rates, and hormone profile) before and after stress. The thickness of chlorenchyma was initially larger in M28 plants, but was significantly reduced after heat stress, while increased in M18 plants. Irrespective of their origin, when these plants were subjected to a heat treatment, relative water content (RWC) and photosynthetic carbon assimilation rates were not significantly affected, although M18 plants increased net photosynthesis rate after 10 days recovery (tR). M18 plants showed proline contents that increased dramatically (2.4-fold) when subjected to heat stress, while proline contents remained unaffected in M23 and M28 plants. Heat stress significantly increased abscisic acid (ABA) content in the needles of maritime pine plants (1.4-, 3.6- and 1.9-fold in M18, M23, and M28 plants, respectively), while indole-3-acetic acid content only increased in needles from M23 plants. After the heat treatment, the total cytokinin contents of needles decreased significantly, particularly in M18 and M28 plants, although levels of active forms (cytokinin bases) did not change in M18 plants. In conclusion, our results suggest that maturation of maritime pine somatic embryos at lower temperature resulted in plants with better performance when subjected to subsequent high temperature stress, as demonstrated by faster and higher proline increase, lower increases in ABA levels, no reduction in active cytokinin, and a better net photosynthesis rate recovery.

Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants.

Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L-1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

Cloning and functional identification of setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1).

Plant somatic embryogenesis receptor-like kinases (SERK), members of leucine-rich repeat receptor-like kinases (LRR-RLKs) subfamily, are widely involved in plant growth, development and innate immunity. In this study, the setaria italica somatic embryogenesis receptor-like kinase1 gene (SiSERK1) was cloned by gateway technology, and transferred into a brasssinosteroid (BR) receptor mutant of Arabidopsis thaliana WS2 (bri1-5). After BL treatment, the transgenic plants could partially restore the phenotype of bri1-5. After Pst DC3000 treatment, the CFU value of SiSERK1 overexpression plant pathogen was between WS2 and bri1-5. Stomatal opening and plant height were also between them. Therefore, it is speculated that SiSERK1 gene is involved in BR signaling pathway and can improve the resistance of bri1-5 to Pst DC3000 through SA and NHP mediated systemic acquired resistance (SAR).

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.