Embryogenic Masses Research Articles

Induction and maturation of somatic embryogenesis in mangaba tree a multipurpose Apocynaceae

This study reported the first results of in vitro somatic embryogenesis induction in Hancornia speciosa Gomes (mangaba tree, Apocynaceae). To induce callus, the TC accession leaf and nodal segments were cultivated in culture medium I (Murashige and Skoog; MS salts, putrescine, 45 µM 2,4-dichlorophenoxyacetic acid; 2,4-D and 22.19 µM 6-benzylaminopurine; BA). For the next stage (multiplication), the callus masses were sub-cultured into a culture medium II (MS salts, 22.62 µM 2,4-D and 22.19 µM BA). After 90 days, embryogenic calluses were inoculated to somatic embryo maturation medium culture III (MS salts, polyethylene glycol; PEG (0 and 2%) and BA (44.39 and 66.58 µM). Pro-embryos and somatic embryos in the maturation stage were assessed after 60 days. The rstatix package from R software with non-parametric tests (p < 0.05) were used to analysis data of experiments I and II. The Tukey’s test (p < 0.05) was performed on the data of experiment III. To induce and multiply embryogenic callus, 45 µM 2,4-D and 22.19 µM BA were efficient. More pro- and somatic embryo development were seen in the nodal explant cultured in the presence of 44.39 and 66.58 µM BA and 2% PEG. Putrescine does not promote the embryogenic masses of callus derived from leaf explants in the TC accession. Keywords: Hancornia speciosa Gomes, micropropagation, somatic embryo.

Plant peptide hormone phytosulfokine promotes embryo development of mass in Pinus massoniana

Pinus massoniana is a critical afforestation and ecological tree species in China. However, the continued existence of this pine is severely threatened by pine wilt disease. Somatic embryogenesis serves as a highly efficient clonal propagation approach. Although significant progress has been made in somatic embryogensis research on P. massoniana, resulting in the successful regeneration of plants, the limited embryogenic potential of improved cell lines and loss of embryogenic properties resulting from prolonged proliferation have posed obstacles to the industrialization of SE production. In this study, we investigated the effect of phytosulfokine on embryo development of cell lines from P. massoniana which lead to a cascade of physicochemical changes. Eight embryogenic cell lines of P. massoniana were used to observe phenotype and cytological changes. Physiological factors and the contents of nutrients and endogenous hormones were measured before and after phytosulfokine addition. We found that PSK promoted a change in the embryogenic mass of P. massoniana, leading to their development from pro-embryogenic mass (PEM)I to PEMII or PEMIII stages of pro-embryos. In addition, PSK accumulated soluble sugar, protein, and starch, and maintained redox homeostasis during cell line proliferation by reducing H2O2 levels. Our findings increase our understanding of how PSK affects somatic embryogensis in P. massoniana, thereby providing a valuable tool for establishing efficient somatic embryogensis systems in conifer species.

Somatic Embryogenesis and Agrobacterium-Mediated Gene Transfer Procedures in Chilean Temperate Japonica Rice Varieties for Precision Breeding

Rice (Oryza sativa) varieties are generated through breeding programs focused on local requirements. In Chile, the southernmost rice producer, rice productivity relies on the use and generation of temperate japonica germplasms, which need to be adapted to the intensifying effects of climate change. Advanced biotechnological tools can contribute to these breeding programs; new technologies associated with precision breeding, including gene editing, rely on procedures such as regeneration and gene transfer. In this study, the local rice varieties Platino, Cuarzo, Esmeralda, and Zafiro were evaluated for somatic embryogenesis potential using a process that involved the combined use of auxins and cytokinins. An auxin-based (2,4-D) general medium (2N6) allowed for the induction of embryogenic masses in all the genotypes. After induction, masses required culturing either in N6R (kinetin; Platino) or N6RN (BAP, kinetin, IBA, and 2,4-D; Cuarzo, Esmeralda, and Zafiro) to yield whole plants using regeneration medium (N6F, no hormone). The sprouting rates indicated Platino as the most responsive genotype; for this reason, this variety was evaluated for gene transfer. Fifteen-day-old embryo masses were assayed for Agrobacterium-mediated transformation using the bacterial strain EHA105 harboring pFLC-Myb/HPT/GFP, a modified T-DNA vector harboring a geminivirus-derived replicon. The vector included the green fluorescent protein reporter gene, allowing for continuous traceability. Reporter mRNA was produced as early as 3 d after agroinfiltration, and stable expression of the protein was observed along the complete process. These achievements enable further biotechnological steps in these and other genotypes from our breeding program.

DNA demethylation by transitory 5‐azacytidine treatment improves somatic embryogenesis yield for regeneration and breeding of cork oak

AbstractSomatic embryogenesis (SE) is a crucial biotechnological tool for large‐scale mass propagation of selected material, genetic transformation and breeding, with many advantages for forest tree improvement. However, the application of SE in forest species is limited because of their recalcitrance. SE is also a valuable system for studying cell reprogramming, acquisition of totipotency and embryo development. Increasing evidence reveals that epigenetic reprogramming takes place during SE through DNA methylation, although there is scarce information on forest species. In this work, we have evaluated DNA methylation dynamics during SE and the effects of the DNA demethylating agent 5‐azacytidine (AzaC) in cork oak. After induction and early stages of SE, a reduced DNA methylation level is observed, followed by an increase of methylation during embryo differentiation. These changes in DNA methylation during SE progression were associated with expression profiles of DNA methyltransferase genes QsMET1, QsDRM2 and QsCMT3, and DNA demethylase QsDME‐like. Treatment with AzaC reduced DNA methylation, promoted SE induction rate and proembryogenic masses proliferation, and induced the expression of the SE marker gene QsSERK1‐like. However, continuous AzaC treatment hindered embryo differentiation, suggesting that DNA methylation is needed for further embryo development. Interestingly, AzaC removal from the culture medium of embryogenic masses restored embryo development and led to a significant increase in somatic embryo production compared with untreated cultures. These findings open new possibilities using transitory treatments with small molecule epigenetic modulators, as AzaC, to enhance SE yields for forestry breeding programs.

English

Androgenesis is reported in a few tree crops such as citrus and papaya; however, there is little information available about guava. Different modified Chu (N6) media and cold pretreatments of floral buds were tested to induce embryogenic calli in the anthers of two white flesh guava cultivars, ‘Round’ and ‘Pyriform’. Four modified Chu (N6) media formulations, i.e., M1 (400 mgL-1 casein hydrolysate, 200 mgL-1 glutamine, 0.2 mgL-1NAA, 1.0 mgL-1 KIN, 0.5 mgL-1 6-BAP, 0.5 mgL-1 zeatin and 5% coconut water), M2 (400 mgL-1 casein hydrolysate, 200 mgL-1 glutamine, 0.2 mgL-1NAA, 1.0 mgL-1 KIN, 0.5 mgL-1 6-BAP), M10 (0.5 mgL-1 2,4-D), and M11 (0.5 mgL-1 2,4-D and 50 mgL-1 PVP) enhanced anther swelling and callus formation. Genotypic variability was found in pollen viability, phenolic exudation, and anther tissue browning after cold pretreatment for 0-72 hrs. intervals. Anthers of ‘Round’ showed more pollen viability across cold pretreatment intervals with less phenolic exudations and tissue browning than ‘Pyriform’. On both M10 and M11 media, anthers with uni-nucleate microspores developed embryogenic calli; however, anthers of ‘Pyriform’ resulted in more calli induction (69.58%) on M10 media, whereas anthers of ‘Round’ developed more calli (66.60%) on M11 media. The proliferating calli in white flesh ‘Round’ and ‘Pyriform’ cultivars and pink flesh ‘Round’ cultivar formed more embryogenic masses upon transfer to growth hormone-free Chu (N6) media under light conditions; however, embryos showed delayed maturity with limited germination. More genotypes shall be explored to enhance embryo germination, and embryogenic media shall be further fine-tuned. This study provides the foundation for the generation of homozygous lines in guava using androgenesis

Calcium dynamics and modulation in carrot somatic embryogenesis.

Free calcium (Ca2+) is a pivotal player in different in vivo and in vitro morphogenic processes. In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca2+ sensors. With them, Ca2+ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca2+ levels (CaCl2, ionophore A23187, EGTA), to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca2+ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca2+ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca2+ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca2+-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca2+ or calmodulin levels negatively affect the efficiency of the process.

High temperature and water deficit cause epigenetic changes in somatic plants of Pinus radiata D. Don

Current climate changes imply an imminent risk for forest species. In this context, somatic embryogenesis is a valuable tool to study the response of plants to different abiotic stresses. Based on this, we applied a high-temperature regime (50 °C, 5 min) during the maturation of Pinus radiata D. Don embryogenic masses in order to evaluate the development of an epigenetic memory months later. Therefore, somatic plants (SP) resulting from somatic embryos (ses) maturated at control temperature and cultivated in a greenhouse were submitted to heat stress (40 °C, 2 h, 10 days; 23 °C, 10 days) or at a control temperature (23 °C, 20 days); while another 20 SP resulting from ses maturated in the two temperature regimes and cultivated in the greenhouse were submitted to drought stress or weekly irrigated. All plants were evaluated for relative water content, water potential, electrolyte leakage, stomatal conductance, transpiration, methylation (5-mC) and hydroxymethylation (5-hmC) levels. The results showed that the SP obtained from ses maturated at 50 °C showed an adaptation to drought stress based on water potential and transpiration. Furthermore, SP kept under heat stress in a greenhouse showed lower 5-hmC levels than SP kept at 23 °C. Furthermore, the 5-hmC and 5-hmC/5-mC ratio showed a significantly negative correlation with changes in water potential; and a significantly negative correlation was observed between the levels of stomatal conductance and 5-mC. We conclude that the manipulation of conditions during the maturation process in somatic embryogenesis modulates the physiological characteristics of the SP obtained. Application of high temperatures during maturation and hardening of somatic plants provoked drought adaptation and epigenetic changes.

Embryogenic cultures and somatic embryos development from mature seeds of jabuticaba (Plinia cauliflora (Mart.) Kausel)

Plinia cauliflora is an important Brazilian species that produces highly appreciated fruits, with a great potential of commercialization. However, the high cost of seedlings is a bottleneck for the expansion of commercial orchards. The present study aimed to investigate somatic embryogenesis as a propagation method for P. cauliflora using seeds as explants. To induce embryogenic mass (EM) and somatic embryo (SE) development we evaluated the supplementation of culture medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), combined or not with activated charcoal (AC). For the embryo maturation, we investigated the effects of AC, polyethylene glycol (PEG), Gelzan®, 6-benzylaminopurine and gibberellin supplementation. For the EM induction, the best results were obtained in MS culture medium supplemented with 300 μM 2,4-D and 1 g L-1 AC. During the first maturation phase, the supplementation of 30 g L-1 PEG improved the somatic embryo formation at the torpedo and cotyledonary stages, whereas the maturation treatments did not result in the conversion of the embryos into plantlets. The anatomical analysis showed that the 2,4-D presence for 60 days may have been deleterious for embryonic development. These results represent the first report of P. cauliflora somatic embryogenesis and its feasibility for mass propagation.

EFFECT OF 2,4-D AND NAA ON SOMATIC EMBRYOGENESIS FROM APICAL PORTION OF GROUNDNUT (ARACHIS HYPOGAEA L.)

Somatic embryogenesis was carried out epicotyl portion of the mature embryo/apical portion. The somatic embryo induction medium containing 2,4-D or NAA (10.0 to 50.0 mg/l). Of the two concentrations tested 2,4-D (30.0mg/l) recorded the highest percentage of response followed by NAA (30.0mg/l). But the highest number of somatic embryo were recorded in 30.0mg/l of 2,4-D followed by NAA. The apical portion of the mature embryo formed direct embryos without any intervention of callus. The maximum percentage of embryogenic cultures were noticed in 30.0mg/l of 2,4-D followed by NAA at 30.0mg/l. for the differentiation of somatic embryos, the embryogenic masses were transferred to medium without any growth regulator. The maximum number of somatic embryos per culture was recorded in 30 mg/l of 2,4-D followed by 30.0 mg/l of NAA. Keywords: Arachis hypogaea L.,Somatic Embryogenesis, 2,4-D and NAA

Optimization of Different Factors for Initiation of Somatic Embryogenesis in Suspension Cultures in Sandalwood (Santalum album L.)

Santalum album (L.) is a prized tropical tree species of high therapeutic and industrial importance. The wood of these naturally grown plants is extensively harvested to acquire therapeutically important metabolite santalol and be used for additional functions such as in wood statuette industries. Due to high demand, it is crucial to maintain a sufficient plant population. An easy protocol for establishing cell suspension culture initiated from the loose embryogenic callus mass of sandalwood was realized by shifting 6–8-week-old morphogenic calli acquired from the mature embryonic axis and cotyledon explant cultures in fluid media. The asynchronous embryogenic cultures were sloughed with clumps of flourishing cell clumps and embryos of various progressive phases along with diffident non-embryogenic tissues. The frequency of embryo proliferation was evidenced to determinethe expansion pace of embryogenic masses under diverse conditions. The intonation of initiation and creation of cell suspension was under the directive of the influence of exogenous plant growth regulators amended in the nutrient medium at different concentrations and combinations. Maximum relative growth rate (386%) and clumps/embryoids in elevated integers (321.44) were accomplished on MS nutrient medium fortified with 2.0 mg L−1 2,4-D in association with 0.5 mg L−1 BA and 30.0 g L−1 sucrose raised from mature embryonic axis-derived calli. Plantlet regeneration in higher frequency (84.43%) was evidenced on MS medium amended with 1.0 mg L−1 each of TDZ and GA3 in conjunction with 0.5 mg L−1 NAA and 20.0 g L−1 sucrose. Mature embryonic axis-derived calli were found to be constantly better than mature cotyledon-derived calli for raising profitable and reproducible cell suspension cultures. Regenerants displayed normal growth and morphology and were founded successfully in the external environment after hardening.

Priming Maritime Pine Megagametophytes during Somatic Embryogenesis Improved Plant Adaptation to Heat Stress

In the context of global climate change, forest tree research should be addressed to provide genotypes with increased resilience to high temperature events. These improved plants can be obtained by heat priming during somatic embryogenesis (SE), which would produce an epigenetic-mediated transgenerational memory. Thereby, we applied 37 °C or 50 °C to maritime pine (Pinus pinaster) megagametophytes and the obtained embryogenic masses went through the subsequent SE phases to produce plants that were further subjected to heat stress conditions. A putative transcription factor WRKY11 was upregulated in priming-derived embryonal masses, and also in the regenerated P37 and P50 plants, suggesting its role in establishing an epigenetic memory in this plant species. In vitro-grown P50 plants also showed higher cytokinin content and SOD upregulation, which points to a better responsiveness to heat stress. Heat exposure of two-year-old maritime pine plants induced upregulation of HSP70 in those derived from primed embryogenic masses, that also showed better osmotic adjustment and higher increases in chlorophyll, soluble sugars and starch contents. Moreover, ϕPSII of P50 plants was less affected by heat exposure. Thus, our results suggest that priming at 50 °C at the SE induction phase is a promising strategy to improve heat resilience in maritime pine.

Indole acetic acid (IAA) producing endophytic bacteria on direct somatic embryogenesis and plant regeneration of Exacum travancoricum Bedd.

Endophytic bacteria were isolated from Withania somnifera (L.) Dunal and their potentials were explored through the in vitro plant regeneration techniques. Initially, indole acetic acid (IAA), phosphate solubilization and siderophore producing isolates were screened out. These isolates were identified using molecular tools like 16s rRNA gene sequences namely Bacillus pumilus AS02, Sphingobacterium thalpophilum AS34, Pseudomonas aeruginosa AS36, Agrobacterium tumefaciens AS38 and Enterobacter aerogenes AS75. Further, the regeneration efficacy of IAA producing endophytic bacteria has been analysed on an endangered plant—Exacum travancoricum Bedd. The leaf and inter-node explants of E. travancoricum with or without bacterial infection were tested on Murashige and Skoog’s (MS) medium encompassing 6-benzyladenine (BA; 2.0 mg L− 1) and l-tryptophan (0.2%; w/v). Somatic embryo induction was observed on both explants. The leaf explants infected with S. thalpophilum AS34, P. aeruginosa AS36 and E. aerogenes AS75 exhibited highest percentage of somatic embryo induction (93.3%) compared with the inter-node explants (86.6%). The explants without bacterial infection (control) and the presence of exogenous IAA exhibited lower level of response (88.8%, leaf and 75.5%, inter-node). Histological analysis revealed the appearance of globular embryogenic masses on epidermal, sub-epidermal regions of both explants. The development of torpedo, cotyledon stage and protoderm formation without the vascular connection from explants confirmed direct somatic embryogenesis. This was confirmed with scanning electron microscopy. Sub-culturing of cotyledon somatic embryos in MS liquid medium without growth regulators was performed to obtain well developed shoots with roots.

In vitro callus induction and development of Vernonia condensata Baker with embryogenic potential

ABSTRACT Vernonia condensata Baker has been traditionally used in folk medicine for the treatment of several inflammatory and infectious processes. Overexploitation of this plant species has drastically reduced its population in its natural habitat (Cerrado). Therefore, tissue culture tools, such as somatic embryogenesis, can be used as an alternative method for rapid and large-scale plant regeneration. The objectives of this study were to induce callogenesis in Vernonia condensata from different types of explants and to evaluate the structural aspects of the development of pro-embryogenic masses of this species by means of histological analyses. The formation of calli was induced from leaf explants and internodal segments, which were inoculated in EME medium supplemented with 50 g L-1 sucrose, 0.5 g L-1 malt extract and 2.68 μM NAA, plus varying concentrations of BAP (0.00, 2.22, 4.44 or 8.88 μM). After 40 days, the following morphogenetic traits were evaluated: intensity of callus formation, intensity of oxidation, callus texture, and morphogenesis. The calli with embryogenic masses were analyzed by light and scanning electron microscopy. Both types of explants were responsive regarding callogenesis, with the BAP concentration of 4.44 μM promoting the formation of friable calli associated with a larger percentage of calli with embryogenic masses. Cells from leaf explants and internodal segments were able to dedifferentiate and change into embryonic structures.

In silico and in vivo analysis of ABI3 and VAL2 genes during somatic embryogenesis of Coffea arabica: competence acquisition and developmental marker genes

The employment of biotechnology-based approaches such as somatic embryogenesis has been applied to several plants including Coffea sp. Despite the economic importance of this genus, few information about the role of key regulatory genes in somatic embryogenesis in coffee is available. This work provides information about ABI3 and VAL2 genes performance by RT-qPCR during indirect somatic embryogenesis of Coffea arabica. To achieve this, bioinformatics analysis was performed to identify the genes of the B3 superfamily in the coffee genome. The cell suspensions lines presented similar histological and regeneration patterns, yielding of up to 6.6 embryos per 1 mg of embryogenic aggregates at 7 months. We have identified possible orthologs for VAL2 (Cc06g00410) and ABI3 (Cc01g17380) as well as the other members belonging to superfamily B3. The CaABI3 expression was higher in dedifferentiated competent cells for somatic embryogenesis as compared with non-embryogenic calli. Whereas the expression of VAL2 gene is more active in cotyledonary embryos and plantlets, showing its clear performance in the embryogenesis late stages. The present study suggests that CaABI3 gene could be potentially used as a biomarker for embryogenic process improvement. The good plantlets development obtained from the protocol used may be a reflection of the high expression of CaVAL2 in cotyledonary embryos and plantlets. The activity of CaABI3 is correlated to embryogenic potential with highly expressed in embryogenic masses and expression of the VAL2 gene is increased at the end of the embryogenic process.

New Approaches to Optimize Somatic Embryogenesis in Maritime Pine.

Maritime pine (Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availability during SE process on the production of plants. A strong maternal effect on the embryogenic potential of maritime pine megagametophytes was observed in our experiments using half-sib and full-sib progenies, while paternal effect was almost undetectable. Besides, it seems possible to improve somatic embryo production of maritime pine megagametophytes by adjusting optimal temperature throughout the process: 28°C during induction and proliferation, and 23°C during the maturation phase. Using induction and proliferation media with reduced water availability (6 g/L Gelrite) can also increase embryo production. Since other limitation of maritime pine SE is culture decline of embryogenic masses (EMs), that reduces embryo yield and germination, we assessed the profile of ABA and IAA and the expression of two embryogenesis-related genes (LEC1 and WOX2) during maturation of EMs of two morphotypes that differed in their maturation capability. Spiky morphotype (SK), with high maturation capability, had a steady increase in both hormones along the 12 weeks of the maturation, whereas ABA content in smooth morphotype picked at the 4th week and dropped. EMs with this morphotype also had a higher IAA content at the beginning of the maturation. A decrease of LEC1 and WOX2 gene expression over the course of embryo development was found to be characteristic of the SK with high maturation capability.

5-Azacitidine Induces Cell Death in a Tissue Culture of Brachypodium distachyon.

Morphological and histological observations revealed that, at a concentration of 50 µM, 5-azacitidine (5-azaC) totally inhibited the induction of embryogenic masses (EM), while the cultivation of explants (zygotic embryos; ZEs) in the presence of 5 µM of 5-azaC led to the formation of a callus with EM in 10% of the cases. Transmission electron microscopy (TEM) analyzes revealed the presence of the morphological and ultrastructural features that are typical for the vacuolar type of cell death in the callus cells that were treated. A TUNEL assay confirmed the presence of DNA double-strand breaks for the callus cells that had been treated with both 5 and 50 µM 5-azaC concentrations. Analysis of the gene expression of selected cell death markers demonstrated a reduced expression of metacaspase, protein executer 1 (EX1), and thioredoxin (TRX) in the callus cells that had been treated compared to the control culture. The strongest increase in the gene activity was characteristic for glutathione S-transferase (GST). Our studies also included an analysis of the distribution of some arabinogalactan proteins (AGPs) and extensin epitopes, which can be used as markers of cells that are undergoing death in a Brachypodium distachyon tissue culture.

Development of Somatic Embryo Maturation and Growing Techniques of Norway Spruce Emblings towards Large-Scale Field Testing

The possibility to utilize non-additive genetic gain in planting stock has increased the interest towards vegetative propagation. In Finland, the increased planting of Norway spruce combined with fluctuant seed yields has resulted in shortages of improved regeneration material. Somatic embryogenesis is an attractive method to rapidly facilitate breeding results, not in the least, because juvenile propagation material can be cryostored for decades. Further development of technology for the somatic embryogenesis of Norway spruce is essential, as the high cost of somatic embryo plants (emblings) limits deployment. We examined the effects of maturation media varying in abscisic acid (20, 30 or 60 µM) and polyethylene glycol 4000 (PEG) concentrations, as well as the effect of cryopreservation cycles on embryo production, and the effects of two growing techniques on embling survival and growth. Embryo production and nursery performance of 712 genotypes from 12 full-sib families were evaluated. Most embryos per gram of fresh embryogenic mass (296 ± 31) were obtained by using 30 µM abscisic acid without PEG in the maturation media. Transplanting the emblings into nursery after one-week in vitro germination resulted in 77% survival and the tallest emblings after the first growing season. Genotypes with good production properties were found in all families.

Explant origin and culture media factors drive the somatic embryogenesis response in Acrocomia aculeata (Jacq.) Lodd. ex Mart., an emerging oil crop in the tropics

The explant origin and the factors in the culture medium are responsible for the response to somatic embryogenesis in most plant species, including palms. Acrocomia aculeata is a neotropical palm currently undergoing domestication due to its sustainability and profitability as an alternative oil crop. The attainment of productive cultivars of this palm may be speed up by somatic embryogenesis. Thus, this study sought to refine an existing somatic embryogenesis cloning protocol for A. aculeata and to investigate the possible genetic influence on the in vitro propagation response, using zygotic embryos from 19 matrices or open-pollinated families. The embryos were inoculated in modified induction media. The following characteristics were evaluated in this phase: swollen embryos, percentage of zygotic embryos that formed callus, embryogenic calli and oxidation. Afterwards the resulting pro embryogenic masses were subsequently cultivated in modified multiplication, maturation and gemination media. In those phases the open-pollinated families’ response was measured by the growth of their embryogenic mass and the quantity of regenerated and germinated somatic embryos. The refined micropropagation protocol was successful in establishing embryogenic lines and maintaining their cyclic multiplication for a long period; all without the loss of their potential to maturate and regenerate somatic embryos. In general, the multiplication of pro embryogenic masses was effective even in the lowest dose of Picloram (18 μM). The addition of activated charcoal in the medium favored the maturation of somatic embryos. Plantlets regeneration, although defective in some lineages, was promoted by GA3 (0.55 μM). The zygotic embryos’ responsiveness to somatic embryogenesis varied according to their matrices, indicating that if micropropagation is to be used as a mass propagation technique for the species, the explant́s genotype must be considered.

Strategies for Fast Multiplication and Conservation of Forest Trees by Somatic Embryogenesis and Cryopreservation: a Case Study with Cypress (Cupressus sempervirens L.)

Common cypress (Cupressus sempervirens L.) is one of the most widespread species in the Mediterranean area. It has been traditionally cultivated for its ornamental value, becoming a typical feature of urban and rural landscapes, and high timber quality. In the last 30 years, cypress has been subjected to important breeding programmes, aimed to select clones tolerant to the widespread canker, caused by the pathogenic fungus Seiridium cardinale, leading to various patented varieties today available on the market, as well as for genotypes producing null or low amount of allergenic pollen. Somatic embryogenesis is a suitable in vitro regeneration method for fast cloning of conifer trees, and the cryopreservation of embryogenic callus is a significant tool for the safe long-term conservation of valuable cell lines. Recently, a complete protocol for the production of cypress plants from somatic embryogenesis was developed for the patented clone ‘Mediterraneo’. Here, the coupling of somatic embryogenesis and cryopreservation may offer a superior tool to propagate and maintain selected genotypes of cypress by overcoming repetitive subculturing of selected embryogenic callus lines. For the above, this study aimed to compare different cryopreservation techniques (PVS2-based vitrification and slow cooling) with the ‘Mediterraneo’ embryogenic callus line. Best results were obtained after the optimization of a slow cooling procedure, based on the 30-min treatment of embryogenic masses with a cryoprotective solution containing 180 g l-1 sucrose and 7.5% DMSO, followed by the reduction of the temperature at a rate of -1 °C min-1 up to -40 °C and the subsequent immersion in liquid nitrogen (“two-step freezing”).

Identification of Serk Gene from Bract Derived Embryogenic and Non - Embryogenic Calli of Four Diploid Banana Cultivars from South India

Somatic embryogenesis receptor kinase (SERK) gene is known to be a marker of somatic embryogenesis in several plant species. The present study reported the presence of SERK gene from bract derived embryogenic calli bearing somatic embryos. The analysis of the expression pattern of the SERK gene during embryogenic cell formation and somatic embryogenesis revealed that SERK expression continued during pro embryogenic mass formation. In the present study the amplified product of cDNA from the somatic embryos has molecular size 1459 bp. The non- embryogenic callus also showed the presence of faint bands. In all the samples the amplified product from β - actin primer showed bands of 650 bp with similar intensity in both the embryogenic and non- embryogenic samples.