Single Somatic Embryos Research Articles

From induction to embryo proliferation: improved somatic embryogenesis protocol in grapevine for Italian cultivars and hybrid Vitis rootstocks

Somatic embryogenesis is the most common regeneration method for the application of new genomic techniques like cisgenesis/intragenesis, genome editing, and RNAi. However, some local important genotypes show recalcitrance to this morphogenetic strategy, which represents an obstacle for the application of genetic engineering techniques. Whole flowers, stamens, and pistils of three different Italian Vitis vinifera L. cultivars (Ancellotta, Glera, and Lambrusco Salamino), and four hybrid rootstocks (110 Richter, 17.37, SO4, Star 50) have been tested in several culture media with changing basal salts (NN and MS), different combinations of growth regulators (BAP, 2,4-D, NOA, PIC, and NAA), and gelling agents, to initiate somatic embryogenesis. The formation of embryogenic calli was observed mainly from whole flowers cultured on PIV medium (NN salts, B5 vitamins, 3 g L−1 gelrite, 60 g L−1 sucrose, 8.9 µM BAP, and 4.5 µM 2,4-D), and stamens on MS1 medium (MS salts and vitamins, 7 g L−1 plant agar, 20 g L−1 sucrose, 4.5 µM BAP, and 5 µM 2,4-D), in the cv. Ancellotta, Lambrusco Salamino, and all the rootstocks, except for Star 50, which showed the best embryogenetic response from pistils placed on MS1. In a recalcitrant cv. as Glera, pistils placed on MS medium supplemented with 1 µM BAP, 5 µM 2,4-D, and gelrite as gelling agent, showed the highest percentage of embryogenesis. In addition, a two-step protocol was efficiently optimized for further induction of secondary embryo production for the above-listed grapevine genotypes, which guaranteed the long-term maintenance of embryogenic cultures from clusters or single somatic embryos.

Pengaruh Hormon terhadap Induksi Embrio Somatik Kacapiring (Gardenia jasminoides) dan Potensi Aplikasinya dalam Pembuatan Benih Sintetik

Kacapiring (Gardenia jasminoides) is an ornamental plant that has many uses, as a cut flowers, food ingredients, textile materials, cosmetic raw materials, and containing therapeutic compounds. The high demand for gardenia needs to be balanced with the results of its cultivation. Conventional propagation of gardenias has disadvantages such as a long life cycle, genotypes can differ from the parent, and can carry disease from the parent. Therefore, more efficient gardenia propagation techniques are needed, one of which is by making synthetic seeds. Synthetic seeds can be produced by encapsulating somatic embryos. Somatic embryos are collections of embryonic cells formed from soma cells that have the ability to regenerate into a complete new plant. Hormones are organic materials that occur naturally in plants or are synthetic. Hormones have an effect on plant differentiation and growth. Somatic embryogenesis can be carried out using auxin and cytokinin hormones, because auxins can induce embryogenic callus and cytokinins can initiate the embryogenic process in callus. Synthetic seeds are single somatic embryos covered by a matrix, so that they can be stored for a certain duration of time and can grow into a complete plant in both in vitro and ex vitro conditions. This literature review is to provide an overview based on existing references on the effects of various types of hormones for the induction of gardenia somatic embryos, synthetic seed production methods, and synthetic seed storage temperatures.

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.]).

Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques’ scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines’ anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

Somatic Embryogenesis of Lilium from Microbulb Transverse Thin Cell Layers.

A reliable somatic embryogenesis protocol is a prerequisite for application of other plant biotechniques. Several protocols were reported for genus Lilium, with variable success. Between them, transverse Thin Cell Layers (tTCL) were used efficiently to induce indirect somatic embryogenesis of Lilium. Somatic embryogenesis potential is dependent on the genotype, explant, and culture medium composition, especially as for plant growth regulators and environmental conditions. Usually, the process comprises three phases: embryogenic callus induction, embryogenic callus proliferation and somatic embryo germination. Somatic embryo germination can be achieved in light or dark. In the first case, complete plantlets are formed, with green leaves and pseudobulb in the base. In darkness, microbulbs are formed from single somatic embryos or clusters. A last phase of microbulb enlargement allows plantlets or microbulbs to increase their biomass. These enlarged microbulbs do not need special acclimatization conditions when transferred to soil and quickly produce sturdy plants. This chapter describes a protocol for somatic embryogenesis of Lilium using tTCL from microbulbs.

The development of techniques for tissue culture of mango (Mangifera indica L.) var. Carabao and successful transfer of ex vitro-grafted plants to soil and the field

The mango, a virtually neglected fruit before the advent of nitrate-induced flowering, is now the third leading export fruit in the Philippines, with the ‘Carabao’ mango being the only variety exported. Delay or control of ripening, as well as the ability to program market availability, would further increase the value of mango. Prolonging its shelf life and improving other characteristics can be achieved through biotechnology, requiring a reliable tissue culture regeneration protocol, and a successful transfer-to-soil system. Between 2004 and 2008, immature fruits were collected and cultured in vitro. Primary somatic embryo (SE) induction ranged from 16–100% depending on the strain, collection time, and tree source. A single SE was required to initiate SE proliferation. When subcultured, the SEs proliferated, germinated, and produced plantlets. Initial shoot formation was 8–64%, while succeeding true leaf formation was reduced to 0–36%. In some cultures, abscission and browning were observed, but gas chromatography assays ruled out ethylene as the cause. Regenerated plants survived transfer to soil, but at low percentages. While it took 1 y to develop the tissue culture protocol, it took 3 y to develop the technique for successful transfer of plantlets to soil, and to the field. This is the first report, of a polyembryonic mango, variety Carabao, for which successful tissue culture and field transplantation systems were developed. The technology is equally important as a way to rapidly propagate uniform rootstocks of superior ‘Carabao’ strains or other varieties, for generation of variability, genetic transformation, and for germplasm conservation and exchange.

Genetic transformation of olive somatic embryos through Agrobacterium tumefaciens and regeneration of transgenic plants

Transformed olive plants were regenerated from inoculated somatic embryos with Agrobacterium tumefacience strain GV3101, which carries the plasmid pBI-P5CS containing Arabidopsis thaliana P5CS cDNA, kanamycin marker (npt) gene and uidA reporter gene. Initially, repetitively embryogenic cultures were established from radicles and cotyledonary segments of mature olive zygotic embryos. Single somatic embryos at cotyledon stage were used for transformation. Through repetitive somatic embryogenesis, non-chimer secondary embryos were selected and propagated on kanamycin containing medium. Resistant embryos were converted to plantlets by subjecting them to desiccation. Transformation and P5CS gene expression was confirmed by β-glucuronidase (GUS) assay polymerase chain reaction (PCR) and reverse transcription (RT)-PCR analysis. Key words : Olea europaea, somatic embryogenesis, transformation, β-glucronidase, P5CS gene.

Somatic embryogenesis of two new Panax ginseng cultivars, Yun-Poong and Chun-Poong

Somatic embryogenesis from single cells is important for normal plant regeneration of ginseng. Cotyledon explants from zygotic embryos of two new ginseng cultivars, Chun-Poong and Yun-Poong, produced somatic embryos on Murashige and Skoog (MS) basal medium and MS medium containing growth regulators. The highest frequency of single somatic embryo formation was obtained when cotyledon explants were excised from premature (cultured for 1 day) zygotic embryos (about 6 mm in length) of both cvs. Chun-Poong and Yun-Poong and then cultured on MS medium supplemented with 7% sucrose. The frequency of single somatic embryo formation was strongly enhanced when Chun-Poong cotyledons were subjected to plasmolysis with 0.1–0.5 M sucrose for 24 h and Yun-Poong cotyledons to plasmolysis with 1.0 M sucrose for 24 h and then cultured on MS medium with 2,4-D.

Elimination of Grapevine fanleaf virus from three Vitis vinifera cultivars by somatic embryogenesis

Indirect somatic embryogenesis was tested as a method for eradication of Grapevine fanleaf virus (GFLV) in three grapevine cultivars. Reverse transcriptase-polymerase chain reaction for GFLV detection was performed on tissues sampled at various steps of the embryogenic process: flower explants, embryogenic and non-embryogenic calli, single somatic embryos and regenerated plants. The virus was detected in all tested anthers and ovaries, while only one sample out of 63 regenerated plantlets was positive in the assay. Although GFLV was able to invade embryogenic calli and embryo-derived plantlets, in our experimental conditions GFLV eradication was obtained by somatic embryogenesis alone, without using heat therapy, with a success close to 100%. Assessment of the phytosanitary status of regenerated plants confirmed that GFLV was not detectable in the newly formed leaves 2 years after their transfer to greenhouse conditions.

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l−1 2,4-D and 0.01 mg l−1 kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l−1 promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l−1 kinetin, 0.1 mg l−1 indole-3-butyric acid, and 10 mg l−1 adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.

Detection and Elimination of Viruses in Callus, Somatic Embryos and Regenerated Plantlets of Grapevine

The distribution of some grapevine viruses in flower explants, embryogenic and non-embryogenic calli, single somatic embryos and plants regenerated from embryogenic cultures was investigated by RT-PCR and ELISA. Immature anthers and ovaries of the cultivars Grignolino infected by GRSPaV, GLRaV-1 and GVA, Muller-Thurgau infected by GRSPaV and GLRaV-3 and Bosco infected by GRSPaV were cultivated on media inducing indirect somatic embryogenesis. Viruses were detected both in anthers and ovaries. Four months after culture initiation 65.6% of tested calli were infected by at least one virus; high percentages of virus infection were found in calli originating from ovaries. No virus was detected in calli tested 8 months after culture initiation, as well as in single somatic embryos or in embryo-derived plantlets. Somatic embryogenesis confirmed its effectiveness in eliminating phloem-limited grapevine viruses. Regeneration of RT-PCR negative plantlets occurred even when at least a sector of the callus was still infected: the mechanism whereby somatic embryos are freed of some viruses could be related to the rapid proliferation of embryogenic cells within the callus or to the origin of the embryogenic callus from virus-free cells within the original explant.

A hydrophilic interaction chromatography coupled to a mass spectrometry for the determination of glutathione in plant somatic embryos

An electrospray ionization mass spectrometric (ESI-MS) determination of glutathione (GSH), a sulfur-containing tripeptide (gamma-Glu-Cys-Gly) with regulation and detoxication functions in metabolisms of most living organisms, from nanomolar to micromolar levels is described. A hydrophilic interaction chromatography (HILIC) with an isocratic elution using a mobile phase containing acetonitrile and an aqueous 0.00005% solution of trifluoroacetic acid (60/40%, v/v) was applied for the separation of GSH. The peptide detection was achieved in the presence of L-ascorbic acid which significantly enhanced the signal intensity of the molecular ion GSH [M+H]+ (m/z 308). The calibration curve was linear (R2=0.9995) in the concentration range from 2 nM to 10 microM with a detection limit (LOD, S/N=3) of 0.5 nM. The excellent detection limit, and the excellent selectivity and high reproducibility of this method enabled determination of GSH in a single plant somatic embryo of a Norway spruce (Picea abies). The average amount of GSH in the single somatic embryos (n=18) was 9 pmol per embryo. Owing to our results, it can be supposed that the proposed HILIC/ESI-MS analysis might be used for GSH determination in microscopic cell structures and in single cell analyses.

Nitrilo triacetate increases the rate of single somatic embryos in Asparagus officinalis

Summary A method for the production of single somatic embryos with uniform shape is described in Asparagus officinalis L. Two types of somatic embryos, namely, single embryos and multiple embryos are usually produced. The multiple embryos are aggregations of embryos that did not separate from each other, and these multiple embryos show irregular sizes and shapes. Different concentrations of nitrilo triacetate and application times were studied to increase the number of single embryos. When 100 μmol/L nitrilo triacetate was added to the medium for the production of mature embryos 2 weeks after inoculation of suspension cell clusters, the number of single embryos significantly increased. The somatic embryos produced in the media with nitrilo triacetate successfully developed into normal plants, and there was no significant difference in their conversion frequencies.

Imbibition of white spruce seeds and somatic embryos: A study of morphological changes in an environmental scanning electron microscope and potassium leakage

Potassium leakage and morphological changes during imbibition of white spruce [Picea glauca (Moench) Voss] seeds and somatic embryos were investigated. A single desiccated somatic embryo, a single somatic embryo exposed to a high relative humidity environment for 2 d, and a single dry zygotic embryo leaked similar amounts of potassium over a 120-min period of imbibition in liquid germination medium. A seed without a seed coat leaked two and eight times more potassium than a single whole seed and a single zygotic embryo, respectively. Nearly 50% of the potassium leaked for all tissues was leaked within the first 20 min of imbibition. Exposure of somatic embryos to an environment with high relative humidity resulted in a reduction in the percentage of potassium leaked after 80 and min to levels equivalent to those for zygotic embryos. Using an environmental scanning electron microscope, we found that desiccated somatic embryos and dry zygotic embryos had wrinkled surface cells, with cells in the surface of zygotic embryos being more shrunken in appearance. Imbibition of both types of embryos in water resulted in turgid surface cells after 2 h. Imbibition in liquid germination medium did not cause much hydration of surface cells, which still had wrinkled appearances after 2 h. Finally, imbibition on filter paper on semisolidified germination medium resulted in slower hydration of somatic and zygotic embryos. Cells near the medium appeared hydrated while cotyledon surface cells furthest from the medium resembled cells in desiccated embryos.

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA3) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA3 treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA3 treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos.

Mineral nutrition in white spruce ( Picea glauca [Moench] Voss) seeds and somatic embryos. I. phosphorus, phytic acid, potassium, magnesium, calcium, iron and zinc

The mineral nutrient storage reserves in white spruce ( Picea glauca [Moench] Voss) somatic embryos and various parts of white spruce seeds were investigated. Somatic embryos are embryos produced in tissue culture. A single somatic embryo or zygotic embryo contained similar amounts of P and both types of embryos had similar amounts of the total P present in phytic acid (PA). The large female gametophyte tissue, which is absent in a somatic embryo, contained 86% of the total P and 95% of the total phytic acid-phosphorus (PA-P) in a single seed. The female gametophyte of a single seed contained ≈7 times more P and ≈22 times more PA-P than a single zygotic embryo. Somatic and zygotic embryos had significantly less of their P present in PA than did the female gametophyte tissue, which has over 87% of its P present in PA. A single somatic embryo or zygotic embryo contained similar amounts of K, Mg, Ca, Fe and Zn. The female gametophyte tissue contained 59% of the K, 85% of the Mg, 28% of the Ca, 69% of the Fe, and 65% of the Zn in a single seed. The seed coat of a single seed contained the majority of a seed’s Ca (71%) and also contained significant levels of K, Fe and Zn. On a dry weight (DW) basis, differences were found in the amounts of all the mineral nutrients studied here because somatic embryos are larger than zygotic embryos. Overall, a white spruce somatic embryo has equivalent amounts of all these nutrients as found in a natural white spruce zygotic embryo.

INFLUENCE OF CULTURE CONDITIONS ON 7S GLOBULINS ACCUMULATION IN SOMATIC EMBRYOS OF OIL PALM (ElLAEIS GUINEENSIS JACQ.)

ISHS International Symposium on Biotechnology of Tropical and Subtropical Species Part 2 INFLUENCE OF CULTURE CONDITIONS ON 7S GLOBULINS ACCUMULATION IN SOMATIC EMBRYOS OF OIL PALM (ElLAEIS GUINEENSIS JACQ.)

Accumulation of storage protein and 7S globulins during zygotic and somatic embryo development in Elaeis guineensis

The 7S globulins are the predominant storage proteins in oil palm ( Elaeis guineensis Jacq.) zygotic embryos. The accumulation of these proteins was studied in embryos during seed development and in single somatic embryos cultured in vitro. Antibodies raised against these proteins were used for their detection by western blot and quantification with ELISA. In zygotic embryos, the 7S globulins were deposited mainly between the 14th and the 17th post-anthesis week, corresponding to the end of embryo growth. This represented 10 % of the dry weight and 50 % of soluble proteins. The amount of soluble proteins and 7S globulins in somatic embryos increased rapidly during the early stage of development, but were almost 80 times lower than in zygotic embryos. In somatic embryos, 7S globulins represented 0.3 % of dry weight and 4 % of soluble proteins. After 22 days development, the protein content declined slowly, suggesting a lack of embryo maturation and an early germination.

Plant regeneration from encapsulated somatic embryos of Carica papaya L.

Carica papaya L. (papaya) single somatic embryos (2.0 mm diameter) produced in a high-frequency liquid production system were encapsulated in two different synthetic encapsulation compounds. The frequency of regeneration from encapsulated embryos was significantly affected by (1) the concentration of sodium alginate, (2) the presence or absence of nutrient salts in the capsule, and (3) the duration of exposure to calcium chloride. A 2.5% sodium alginate concentration in a half-strength MS salts base resulted in significantly higher germination frequencies than other treatments. A relatively short (10 min) exposure to CaCl2 provided uniform encapsulation of embryos and the highest frequencies of successful germination (77.5%). Germinated artificial seeds produced normal plantlets.

Establishment of a Plant Regeneration and Genetic Transformation System of Panax ginseng C.A. Meyer

Plant regeneration of ginseng has been known to be difficult, and there are a few reports on plant regeneration of ginseng via somatic embryogenesis. In vitro flowering has, however, been one of the major drawbacks in these regeneration systems in which BA and GA3 were included in germination and shoot multiplication media. Multiplication of adventitious shoots from a single somatic embryo, abnormal morphology, and vitrified shoots were also observed. All these facts have made successful acclimatization of ginseng plantlets difficult. The purposes of this study were 1) to establish the plant regeneration system via organogenesis, 2) to improve normal plant regeneration via somatic embryogenesis, 3) to improve the efficiency of plant regeneration from protoplast culture, 4) to understand the acclimatization process, 5) to develop effective genetic transformation protocol. Data in relation with all these studies are presented in detail.

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajecarska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l−1 sucrose, 1 g l−1 yeast extract and 0.05 mg l−1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l−1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or β-glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.