Embryogenic Cell Research Articles

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.

Histological studies on somatic embryogenesis in rice (Oryza sativ L.)

Rice (Oryza sativa) embryogenic calli were obtained from mature zygotic embryos culture using two varieties of rice, Sakha 101 and Sakha104 on Murashige and Skoog (1962 ) medium supplemented with 1mg/l, 2mg/l and 3mg/l 2,4- dichlorophenoxy acetic acid. The results indicated that the best medium was that containing 3mg/l. Histological analysis of somatic embryogenesis revealed that after two weeks of culture of explants on the callus induction medium, somatic embryo development began with a cluster of proembryogenic cells in the peripheral region of the calli. The outer cell layer of embryogenic calli consisted of small and isodiametric cells, each with a dense cytoplasm and a prominent nucleus and nucleolus, whereas the inner cell layer is composed of large cells with small nuclei and large vacuoles. These embryogenic cells underwent a series of organized divisions and formed the proembryo with a well-defined protodermis. At 20 days after initiation of culture, these proembryos continued a series of organized divisions and gave rise to globular somatic embryos which are delimited by a well-defined layer of epidermal cells with conspicuous nuclei. These somatic embryos had no apparent vascular connection with the mother tissue and had a suspensor. After ten days of culture on regeneration medium, globular somatic embryos developed into heart-shaped somatic embryos, which consisted of cells with prominent nuclei and dense cytoplasm. After 15 days of culture on regeneration medium torpedo-shaped somatic embryos were observed. Then, after 25 to30 days, it turned into a monocotyledon form.

Fatty acids modulate the potential of somatic embryogenesis in cotton

<p indent="0mm">Callus and embryogenic callus are the key stages of cell fate transformation during plant regeneration. The status of callus and embryogenic callus directly determines the ability of somatic embryogenesis of cotton. Callus cells and embryogenic callus cells (EC) of the highly differentiated material ZM24 and the difficult-to-differentiate material TM-1 were generated and selected for transcriptome and metabolome analysis. The fatty acid extension pathway genes <italic>KCS</italic> and <italic>KCR</italic> showed low expression levels in ZM24 and TM-1 callus and in TM-1 ECs, but showed high expression levels in ZM24 EC, suggesting that the expression levels of <italic>KCS</italic> and <italic>KCR</italic> modulate the potential of callus cells developing into ECs. When we measured the fatty acid content in the 20-day-old ZM24 and TM-1 callus cells, we found that the total fatty acid content and the content of individual fatty acid in TM-1 were significantly higher than those in ZM24, indicating that the differences in callus morphology, callus proliferation, and differentiation ability between TM-1 and ZM24 are correlated with changes in fatty acid content. Overexpression of the <italic>Gh_KCS13</italic> gene inhibits callus formation, confirming that KCS13 negatively regulates callus initiation and proliferation by increasing fatty acid content. Transcription factors related to somatic embryogenesis are predicted to regulate the expression of <italic>Gh_KCS13</italic>, providing potential insights into the regulation of cotton somatic embryogenesis. This study has clarified the function of fatty acids in cotton somatic embryogenesis, laying a foundation for optimizing this process through regulating levels of fatty acids.

Integrative proteomics and phosphoproteomics reveals phosphorylation networks involved in the maintenance and expression of embryogenic competence in sugarcane callus

Plant embryogenic cell culture allows mass propagation and genetic manipulation, but the mechanisms that determine the fate of these totipotent cells in somatic embryos have not yet been elucidated. Here, we performed label-free quantitative proteomics and phosphoproteomics analyses to determine signaling events related to sugarcane somatic embryo differentiation, especially those related to protein phosphorylation. Embryogenic calli were compared at multiplication (EC0, dedifferentiated cells) and after 14 days of maturation (EC14, onset of embryo differentiation). Metabolic pathway analysis showed enriched lysine degradation and starch/sucrose metabolism proteins during multiplication, whereas the differentiation of somatic embryos was found to involve the enrichment of energy metabolism, including the TCA cycle and oxidative phosphorylation. Multiplication-related phosphoproteins were associated with transcriptional regulation, including SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH). The regulation of multiple light harvesting complex photosystem II proteins and phytochrome interacting factor 3-LIKE 5 were predicted to promote bioenergetic metabolism and carbon fixation during the maturation stage. A motif analysis revealed 15 phosphorylation motifs. The [D-pS/T-x-D] motif was overrepresented during somatic embryo differentiation. A protein-protein network analysis predicted interactions among SNF1-related protein kinase 2 (SnRK2), abscisic acid-responsive element-binding factor 2 (ABF2), and KIN10, which indicated the role of these proteins in embryogenic competence. The predicted interactions between TOPLESS (TPL) and histone deacetylase 19 (HD19) may be involved in posttranslational protein regulation during somatic embryo differentiation. These results reveal the protein regulation dynamics of somatic embryogenesis and new players in somatic embryo differentiation, including their predicted phosphorylation motifs and phosphosites.

THE EFFECT OF PH AND SUCROSE ON THE EMBRYOGENIC CELLS GROWTH OF SUGAR CANE (Saccharum officinarum) IN LIQUID CULTURE

&lt;p&gt;&lt;strong&gt;Background. &lt;/strong&gt;Sugarcane (&lt;em&gt;Saccharum officinarum&lt;/em&gt; L.) is one of the important commodities in Indonesia as raw material for sugar production. The constraints for sugarcane cultivation are limited availability and poor quality of seedling. The utilization of liquid culture in tissue culture enables to produce good quality seedlings, free of viruses and diseases, and can generate a large quantity in a short time. Liquid culture can produce embryogenic cells faster since embryogenic callus can develop easily in liquid media. &lt;strong&gt;Objective&lt;/strong&gt;. This study determined the appropriate pH and concentration of sucrose in the liquid culture of embryogenic cells. &lt;strong&gt;Methodology&lt;/strong&gt;. This study was conducted in 3 stages of in vitro culture, which were callus induction, solid media proliferation, and liquid media proliferation that using pH and sucrose treatments. &lt;strong&gt;Result&lt;/strong&gt;. The results showed that pH 6.5 + 3% sucrose (A3B1) produced the highest number of callus with the average callus on the first week 29 callus, the second week 8,33 callus and the third week 4,33 callus and the color did not easily turn to brown with color 5Y 8/2. &lt;strong&gt;Implications&lt;/strong&gt;. The protocol was developed that allows embryogenic callus of sugarcane plants to be obtained in liquid media that can be used for embryogenic callus proliferation. &lt;strong&gt;Conclusions&lt;/strong&gt;. pH 6.5 + sucrose 3% (A3B1) was the best combination treatment of pH and sucrose for sugarcane callus proliferation in liquid culture media, which produced the highest number of calluses and color of callus that not easily turned brown.&lt;/p&gt;

MusaATAF2 like protein, a stress-related transcription factor, induces leaf senescence by regulating chlorophyll catabolism and H2 O2 accumulation.

NAC transcription factors are known for their diverse role in plants. In this study, we have demonstrated the role of MusaATAF2, a banana NAC transcription factor, in leaf senescence. Its expression gets strongly up-regulated during the early stress responses of drought and high salinity exposure and down-regulated under ABA application, which suggests MusaATAF2 is a stress-related NAC transcription factor. To study the role of MusaATAF2 in banana, we have transformed the banana embryogenic cells with MusaATAF2 coding region and generated transgenic banana plants. Overexpression of MusaATAF2 in banana plants caused yellow leaf phenotype under control condition, suggesting its role as a senescence-associated transcription factor. Transgenic banana leaves exhibited low chlorophyll content and high H2 O2 accumulation. Hormone analysis of the leaves demonstrated a higher accumulation of ABA in the transgenic plants than the controls. Transgenic plants overexpressing MusaATAF2 have a higher transcript abundance of two chlorophyll catabolic pathway genes (PAO and HCAR) and lower transcript abundance of ROS scavenging enzymes (TDP, THIO, CAT, APX, and PRXDN) than control. Together, all these analyses indicate that MusaATAF2 induces senescence by inducing chlorophyll degradation and H2 O2 accumulation in banana plants and controls its own expression using an ABA-dependent feedback loop.

Somatic Embryogenesis in Vitis for Genome Editing: Optimization of Protocols for Recalcitrant Genotypes

New Plant Breeding Techniques (NPBTs) protocols have been developed to produce new grape varieties with improved quantitative and qualitative characteristics. Reliable transformation protocols for grapes are based on the generation/induction of embryogenic callus cells that are then transformed. Varieties such as Italia have proven to be very recalcitrant to regeneration via somatic embryogenesis. In this work, the development of a protocol for improved production of embryogenic calluses is described. Two sterilization protocols were tested: (a) a lower active chlorine concentration for a longer time (LS); and (b) a higher chlorine concentration for a shorter time (HS), in combination with the absence or presence of citric acid in the growing substrate in the first growth media. The embryogenic calluses formation in Chardonnay, a cv. with a high embryogenic response, was significantly higher in presence of citric acid in the initial growing substrate regardless of the sterilization protocol. In Aglianico, a cv. with a lower embryogenic response, no significant differences were observed. Instead, in a recalcitrant cv. as Italia, we obtained a 13-fold increase in embryogenic calluses formation performing sterilization of flowers with the HS protocol compared to LS.

Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.).

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

Marker-Assisted Selection for Pollen-Free Somatic Plants of Sugi (Japanese Cedar, Cryptomeria japonica): A Simple and Effective Methodology for Selecting Male-Sterile Mutants With ms1-1 and ms1-2.

Pollen allergy caused by sugi (Japanese cedar, Cryptomeria japonica) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of MS1 itself, selecting individuals with ms1-1 and ms1-2 male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of MS1 male-sterile sugi seedlings.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Polyamine Metabolism in Scots Pine Embryogenic Cells under Potassium Deficiency.

Polyamines (PA) have a protective role in maintaining growth and development in Scots pine during abiotic stresses. In the present study, a controlled liquid Scots pine embryogenic cell culture was used for studying the responses of PA metabolism related to potassium deficiency. The transcription level regulation of PA metabolism led to the accumulation of putrescine (Put). Arginine decarboxylase (ADC) had an increased expression trend under potassium deficiency, whereas spermidine synthase (SPDS) expression decreased. Generally, free spermidine (Spd) and spermine (Spm)/ thermospermine (t-Spm) contents were kept relatively stable, mostly by the downregulation of polyamine oxidase (PAO) expression. The low potassium contents in the culture medium decreased the potassium content of the cells, which inhibited cell mass growth, but did not affect cell viability. The reduced growth was probably caused by repressed metabolic activity and cell division, whereas there were no signs of H2O2-induced oxidative stress or increased cell death. The low intracellular content of K+ decreased the content of Na+. The decrease in the pH of the culture medium indicated that H+ ions were pumped out of the cells. Altogether, our findings emphasize the specific role(s) of Put under potassium deficiency and strict developmental regulation of PA metabolism in Scots pine.

Factors Influencing Somatic Embryo Maturation in Sugi (Japanese Cedar, Cryptomeria japonica (Thunb. ex L.f.) D. Don).

This paper presents the results of several experiments identifying basal salts (BS) contained in maturation medium, polyethylene glycol (PEG) concentration, abscisic acid (ABA) concentration, additional supplementation with potassium chloride (KCl), amino acid (AA) concentration, and proliferation culture medium (PCM) as the main culture factors affecting somatic embryo maturation in sugi (Japanese cedar, Cryptomeria japonica, Cupressaceae). Highly efficient embryo maturation was achieved when embryogenic cell lines (ECLs) were cultured on media supplemented with a combination of PEG, ABA, and AAs. More than 1000 embryos per gram of fresh weight (FW) can be produced on EM maturation medium supplemented with 175 g L−1 PEG, 100 µM ABA, 2 g L−1 glutamine, 1 g L−1 asparagine, and 0.5 g L−1 arginine.

WUSCHEL Overexpression Promotes Callogenesis and Somatic Embryogenesis in Medicago truncatula Gaertn.

The induction of plant somatic embryogenesis is often a limiting step for plant multiplication and genetic manipulation in numerous crops. It depends on multiple signaling developmental processes involving phytohormones and the induction of specific genes. The WUSCHEL gene (WUS) is required for the production of plant embryogenic stem cells. To explore a different approach to induce somatic embryogenesis, we have investigated the effect of the heterologous Arabidopsis WUS gene overexpression under the control of the jasmonate responsive vsp1 promoter on the morphogenic responses of Medicago truncatula explants. WUS expression in leaf explants increased callogenesis and embryogenesis in the absence of growth regulators. Similarly, WUS expression enhanced the embryogenic potential of hairy root fragments. The WUS gene represents thus a promising tool to develop plant growth regulator-free regeneration systems or to improve regeneration and transformation efficiency in recalcitrant crops.

Mitochondrial bioenergetics and enzymatic antioxidant defense differ in Paraná pine cell lines with contrasting embryogenic potential

Araucaria angustifolia is classified as a critically endangered species by the International Union for Conservation of Nature. This threat is worsened by the inefficiency of methods for ex-situ conservation and propagation. In conifers, somatic embryogenesis (SE) associated with cryopreservation is an efficient method to achieve germplasm conservation and mass clonal propagation. However, the efficiency of SE is highly dependent on genotype responsivity to the artificial stimulus used in vitro during cell line proliferation and later during somatic embryo development. In this study, we evaluated the activity of antioxidant enzymes and characterized mitochondrial functions during the proliferation of embryogenic cells of A. angustifolia responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The activities of the antioxidant enzymes GR (EC 1.6.4.2), MDHAR (EC 1.6.5.4), and POX (EC 1.11.1.7) were increased in SE1 culture, while in SE6 culture, only the activity of DHAR (EC 1.8.5.1) was significantly higher. Additionally, SE6 culture presented a higher number of mitochondria, which agreed with the increased rate of oxygen consumption compared to responsive SE1 culture; however, the mitochondrial volume was lower. Although the ATP levels did not differ, the NAD(P)H levels were higher in SE1 cells. NDs, AOX, and UCP were less active in responsive SE1 than in non-responsive cells. Our results show significant differences between SE1 and SE6 embryogenic cells regarding mitochondrial functions and antioxidant enzyme activities, which may be intrinsic to the in vitro proliferation phase of both cell lines, possessing a crucial role for the induction of in vitro maturation process.

Lipid Remodeling Confers Osmotic Stress Tolerance to Embryogenic Cells during Cryopreservation.

Plant species conservation through cryopreservation using plant vitrification solutions (PVS) is based in empiricism and the mechanisms that confer cell integrity are not well understood. Using ESI-MS/MS analysis and quantification, we generated 12 comparative lipidomics datasets for membranes of embryogenic cells (ECs) of Magnolia officinalis during cryogenic treatments. Each step of the complex PVS-based cryoprotocol had a profoundly different impact on membrane lipid composition. Loading treatment (osmoprotection) remodeled the cell membrane by lipid turnover, between increased phosphatidic acid (PA) and phosphatidylglycerol (PG) and decreased phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PA increase likely serves as an intermediate for adjustments in lipid metabolism to desiccation stress. Following PVS treatment, lipid levels increased, including PC and PE, and this effectively counteracted the potential for massive loss of lipid species when cryopreservation was implemented in the absence of cryoprotection. The present detailed cryobiotechnology findings suggest that the remodeling of membrane lipids and attenuation of lipid degradation are critical for the successful use of PVS. As lipid metabolism and composition varies with species, these new insights provide a framework for technology development for the preservation of other species at increasing risk of extinction.

Somatic Embryogenesis Initiation in Sugi (Japanese Cedar, Cryptomeria japonica D. Don): Responses from Male-Fertile, Male-Sterile, and Polycross-Pollinated-Derived Seed Explants.

This study aimed to obtain information from several embryogenic cell (EC) genotypes analyzing the factors that affect somatic embryogenesis (SE) initiation in sugi (Cryptomeria japonica, Cupressaceae) to apply them in the improvement of protocols for efficient induction of embryogenic cell lines (ECLs). The results of several years of experiments including studies on the influence of initial explant, seed collection time, and explant genotype as the main factors affecting SE initiation from male-fertile, male-sterile, and polycross-pollinated-derived seeds are described. Initiation frequencies depending on the plant genotype varied from 1.35 to 57.06%. The best induction efficiency was achieved when seeds were collected on mid-July using the entire megagametophyte as initial explants. The extrusion of ECs started approximately after 2 weeks of culture, and the establishment of ECLs was observed mostly 4 weeks after extrusion on media with or without plant growth regulators (PGRs). Subsequently, induced ECLs were maintained and proliferated on media with PGRs by 2–3-week-interval subculture routines. Although, the initial explant, collection time, and culture condition played important roles in ECL induction, the genotype of the plant material of sugi was the most influential factor in SE initiation.

Genetic transformation of apical meristematic shoots in the banana cultivar ‘Williams’

Bananas and plantains (Musa spp.) are among the most critical socioeconomic crops globally, being a staple food for millions of people in the tropics and an essential component for the export market, including the subtropics. Besides conventional breeding, genetic improvement of bananas and plantains could be performed through genetic engineering and new breeding techniques. Furthermore, plant tissue culture is essential for these technologies, including developing embryogenic cell suspensions and in vitro plants. The transient and stable genetic transformation could be performed from in vitro plants, shortening Musa transgenic lines development compared to genetic transformation while using embryogenic cell suspension. In this study, a genetic transformation protocol was established from banana apical meristems for the ‘Williams’ cultivar (genotype AAA). The protocol was based on the co-cultivation of the explants (whole in vitro plants or bisected meristematic tissues derived from in vitro plants) with Agrobacterium tumefaciens strain LBA4404 harboring two binary vectors denominated pLVCIBE1 (cassette: MabHIPP promoter::luc2::Tnos, P35S::hpt::Tnos) and pLVCIBE2 (cassette: P35S::luc2::Tnos, P35S::hpt::Tnos), independently. The stable genetic transformation was obtained by subculturing in vitro banana plants in selection medium (12.5µg/mL of hygromycin) for 8 weeks from bisected meristematic tissue transformation. Genetic transformation was confirmed in vivo with the use of the luciferase reporter gene system. Furthermore, PCR was performed on DNA extracted from leaves of regenerated transgenic in vitro plants after 8 weeks of selection, confirming stable genetic transformation. Therefore, genetic transformation was achieved in the apical meristematic tissue of in vitro banana plants with co-cultivation of Agrobacterium tumefaciens.

Genome-scale analyses of polyketide synthases in banana: Phylogenetics and expression profiling forecast their candidacy in specialized metabolism.

Plant type III polyketide synthases (PKSs) are associated with various functions in plant growth, development and defense by providing a multitude of polyketide scaffolds for diverse specialized metabolic pathways (SMPs). To decipher banana PKSs involved in specialized metabolism, genome-wide comparative analyses were conducted with A (Musa acuminata) and B (Musa balbisiana) genomes of banana. Both genomes retained eight chalcone synthases (CHSs), seven curcumin synthases (CURSs), three diketidyl-CoA synthases (DCSs) and one anther specific CHS (ASC). Segmental (42%) and tandem (37%) duplication events majorly flourished the banana PKS family. Six of 19 PKSs of A genome (designated as MaPKSs) showed relatively a higher expression in the root, corm, sheath, leaf and embryogenic cell suspension (ECS) of banana. To determine the defense response of MaPKSs and to highlight their candidacy in various SMPs, expression profiling was conducted by qPCR in ECSs treated with 100/200μM of jasmonic acid (JA) and salicylic acid (SA) at 24/48h. Maximum and subordinate expression induction of MaPKSs was apparent respectively against JA and SA treatments. Notably, most MaPKSs achieved their peak expression within 24h of JA and the total flavonoid content was reached maximum within 24h of JA/SA elicitations. Considering the homology, phylogeny, and expression levels in each analyzed sample (n=13), three CHSs, three DCSs along with three CURSs and one ASC were selected as most promising candidates respectively for flavonoids, phenylphenalenones and sporopollenin biosynthesis in banana. Our findings provide a first-line resource to disclose the functions of banana PKSs involved in distinct SMPs.

Secondary Somatic Embryogenesis in Centaurium erythraea Rafn.

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU). A medium containing 0.1 mgL−1 2,4-D and 0.25 mgL−1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL−1 2,4-D and 0.5 mgL−1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury—an emerging model in developmental biology.