Somatic embryogenesis by liquid culture of epidermal layers in sunflower: from genetic control to cell development

Embryos were obtained using liquid medium culture of sunflower hypocotyl epidermis layers according to the Pélissier etal. (1990) method. In the present work we identified genetic factors controlling somatic embryogenesis and we evidenced the role of ionic channels in embryogenic tissues. Two traits, the number of embryogenic explants (EE) and the number of embryos (EM) were scored in 74 recombinant inbred lines (RILs) from a cross between lines PAC-2 and RHA-266. Analysis of variance indicated the existence of highly significant differences among the parental genotypes and their RILs. Heritability for the somatic embryogenesis traits studied were high (0.64 for EE and 0.77 for EM). Four quantitative trait loci (QTLs) for EE and seven for EM were detected using composite interval mapping. The QTLs for EE explained 48% of the phenotypic variation while the QTLs for EM explained about 89% of the variation, thus revealing several genomic regions related to somatic embryogenesis control in sunflower. In order to study the distribution of ion channels in somatic embryos as compared to zygotic ones, we used a fluorescent-labelled phenylalkylamine, DM-Bodipy PAA, as a probe. Fluorescence labelling was determined by confocal microscopy. The probe intensively labelled the protoderm and epidermis cells in both zygotic and somatic embryos. Callus exhibited labelling on sites where somatic embryos developed. Considering that the location of phenylalkylamine (PAA) binding sites is related to the distribution of ion channels, the high intensity in the protoderm and epidermis of embryos, point to similar properties and functions and their key role in embryo development.     

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

Summary A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (–)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.Abbreviations PAA phenylalkylamine - DHP dihydropyridine - FDA fluorescein diacetate     

Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium

Cotyledon explants of Korean ginseng (Panax ginseng C. A. Meyer) produced somatic embryos directly on growth regulator-free medium. Somatic embryos developed as either multiple or single-state forms, depending on the degree of maturity of the cotyledons. Cotyledon explants from midmature zygotic embryos formed multiple embryos, while cotyledons from fully mature zygotic embryos formed single embryos. Somatic single embryos regenerated into normal plantlets with both roots and shoots, while multiple embryos did not produce roots but regenerated only into multiple shoots. In full-strength MS basal medium, the root growth of plantlets derived from single embryos was weak compared to that of shoots. Deletion of ammonium nitrate from the MS medium promoted the root growth of the plantlets. The ginseng plants with well-developed shoots and roots regenerated from single embryos were successfully acclimatized in a greenhouse when they were planted in soil. Received: 19 July 1997 / Revision received: 6 October 1997 / Accepted: 3 October 1997     

Rapid propagation of Eleutherococcus senticosus via direct somatic embryogenesis from explants of seedlings

Explants from three different parts (cotyledon, hypocotyl or root) of one week-old seedlings of Eleutherococcus senticosus were cultured on Murashige and Skoog (MS) medium with 1.0 mg l^-1 2,4-D. Somatic embryos were formed directly from the surfaces of explants. The frequency of direct somatic embryo formation was the highest in the hypocotyl segments (75%) as compared to cotyledon (56%) or root segments (12%). When hypocotyl explants from 3 different stages of seedlings (zero, one or three week-old) were cultured on MS medium with 1.0 mg l^-1 2,4-D, the frequency of somatic embryo formation rapidly declined as the zygotic embryos germinated. However most somatic embryos (93%) from explants of zygotic embryos developed as fused state (multiple embryo), whereas somatic embryos (over 89%) from more developed seedlings developed into single state (single embryo). Single embryos germinated and regenerated into plantlets with both shoots and roots, while multiple embryos only regenerated into only multiple shoots. Plantlets that regenerated from single embryos of E. senticosus were acclimatized in a greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dynamics of morphological and anatomical changes in leaf tissues of an interspecific hybrid of oil palm during acquisition and development of somatic embryogenesis

Oil palm is an economically important plant species due to its high oil production per unit area. Large-scale clonal propagation of the species’s elite specimens is only possible through somatic embryogenesis, although methodology is partially still unknown and insufficiently understood. Current study characterizes in morphological and anatomical terms the acquisition and development stages of somatic embryogenesis of the oil palm’s immature leaves. The respective embryogenic stages were analyzed and characterized: immature leaves (initial explants); leaves with calli formation; leaves which failed to respond to calli formation; leaves with formation of root structures; primary calli; primary calli with differentiation of embryogenic calli; embryogenic calli; pro-embryogenic calli; calli with differentiated somatic embryos; somatic embryos at globular and torpedo stage; and mature fruit zygotic embryos. Cell masses emerged after approximately 60 days of cultivation through the proliferation of cells associated to initial explants´ vascular bundles. Consequently, the formation of two different types of calli was identified, namely, primary and embryogenic, respectively consisting partially and completely of meristematic cell clusters. After 420 days of cultivation, the propagules formed somatic embryos with no connection to source tissues, initially composed (globular stage) of a very characteristic ground meristem and protoderm. After 480 days of cultivation, as the cultures matured (torpedo stage), procambial strands, a structural characteristic also observed in mature zygotic embryos, were reported. The results provide an in-depth understanding of somatic embryogenesis of immature leaves of oil palm. Further, current analysis develops morphological markers at different stages of development obtained during the process.     

Vacuolar accumulation of acridine orange and neutral red in zygotic and somatic embryos of carrot (Daucus carota L.)

Summary The accumulation of neutral red and acridine orange, to indicate differences in vacuolar pH, was studied during embryogenesis of carrot. Neutral red accumulated barely in proembryogenic masses, but was present conspicuously in globular-shaped somatic embryos. From the late globular to the torpedo-shaped stage, it was mainly found in the root side of the somatic embryo. Here, neutral red was predominantly present in large dark-red to purple stained vesicles. In the cotyledons neutral red was found in small orange vesicles. In zygotic embryos of carrot, the dye was uniformly distributed with no specific localization in organelles. During germination, however, neutral red accumulated mainly in regions in the root side and the hypocotyl of the germling. Acridine orange was dispersed erratically in proembryogenic masses with a great variety in intensity. It was quite obviously present in early stages of somatic embryogenesis and restricted to the root side in late globular to torpedo-shaped embryos. Confocal images revealed the vacuolar presence of the fluorescence and the predominant presence in the protoderm. During germination of zygotic embryos the signal changed from uniform to localized, with sharp borders between fluorescent and non-fluorescent regions. Two to three days after the beginning of germination, acridine orange accumulated preferentially in the root tip of the germling. Differences between somatic and zygotic embryos and similarities between somatic embryogenesis and zygotic embryo germination are discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pH_c cytosolic pH - pH_e extracellular pH - pH_v vacuolar pH     

Improvement of English walnut somatic embryo germination and conversion by desiccation treatments and plantlet development by lower medium salts

Summary Well-developed somatic embryos were selected from a repetivively somatic embryo line derived from embryonic axes of immature zygotic embryos of English walnut ‘No. 120’ (Juglans regia L.) for germination and conversion studies. In germinating dishes, somatic embryos germinated into only shoots, only roots, or both shoots and roots. Without any pretreatment, 28% somatic embryos germinated, while those treated with 2.5–5.0 mg 1⁻¹ (7.2–14.4 μmol) gibberellic acid (GA₃) germinated at 25–28% and those receiving a cold treatment of 2–3 mo. at 3–4°C germinated at 30–43%. However, only 4–19% of the germinating embryos showed both shoots and roots. Treated with desiccation, either with CaCl₂·6H₂O or Ca(NO₃)₂·4H₂O at 20°C in the dark for 3 d, somatic embryos germinated at 85–91%, 57–69% of which had both shoots and roots. Treatment with 2 mo. cold storage in combination with desiccation using Ca(NO₃)₂·4H₂O resulted in 92% of somatic embryos germinating, 70% of which showed both shoots and roots. No significant differences were observed between solid and liquid germination media. After transferring the germinating embryos to plantlet development media, 52–63% of those with both shoots and roots developed into plantlets while 11% with only shoots or 9% with only roots converted into plantlets. Plantlet development was improved by using lower medium salts and sucrose concentrations. The addition of activated charcoal enhanced root development, particularly root branching. Of 131 plants transplanted, 91 plants were acclimatized to a greenhouse.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis>

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm⁻³ kinetin and 0.2, 0.5 mg dm⁻³ indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.     

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

Summary 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.     

The ability of Prunus avium× P. pseudocerasus `Colt' to form somatic embryos in vitro contrasts with the recalcitrance of P. avium

Somatic embryos were induced on roots excised from in vitro plants of Prunus avium× pseudocerasus `Colt'. On medium containing 6-benzylamino purine (BAP, 1.5 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D, 15 μM), a mean of 25 (s.e. ± 2.0) somatic embryos were produced on intact root systems and 15 (s.e. ± 1.7) on roots systems cut into 10 mm pieces. Most somatic embryos were formed directly on intact roots and indirectly (from callus) on sectioned roots. A mean of 2.5 (s.e. ± 0.25) secondary embryos per primary embryo were formed directly on primary embryos after they were transferred to medium containing BAP (1.5 μM), indole-3-butyric acid (10 μM) and 2,4-D (5 μM). After transfer to a medium containing BAP (2 μM) and gibberellic acid (GA<sub>3</sub>, 3 μM), shoots developed in 75% (s.e. ± 7.3) of the embryos. Somatic embryos were not induced on explants of in vitro roots or shoots of P. avium, and were induced infrequently on zygotic embryos, although a wide range of media were tested. Possible reasons for the contrasting embryogenic ability of `Colt' and P. avium are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1' in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1'. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Immunodetection and immunolocalization of globulin storage proteins during zygotic and somatic embryo development in Zea mays

Globulins (GLB) are storage proteins that accumulate to high levels during zygotic embryo development of Zea mays L. We visualized the distribution of GLB during zygotic embryo development by immunolabelling of polyethylene glycol sections with a GLB-specific antiserum and a fluorescent secondary antibody. In sections of embryos at 10 days after pollimation (DAP), GLB were detected in the scutellar node only. Sections of embryos of 17 DAP showed, besides the presence of GLB in the scutellar node, the presence of a low amount of GLB in the coleoptile and the leaf primordia. In 30-DAP embryos GLB were localized in the root, the coleorhiza, the leaf primordia, the coleoptile and in all cells of the scutellum with the exception of the epidermis and the pro-vascular tissues. The subcellular location of GLB was visualized by immunolabelling of ultrathin sections with anti-GLB and a gold-conjugated secondary antibody. Scutellum cells and root cortex cells of 30-DAP embryos were packed with protein storage vacuoles (PSV), which differed in electron density. GLB were either evenly distributed throughout the PSV or were localized in electron-dense inclusions within the PSV. SDS-PAGE and immunoblot analysis of total protein extracts indicated the presence of a low amount of the GLB1 processing intermediate proGLB1'in globular as well as mature somatic embryos. After maturation on an ABA-containing medium, somatic embryos showed the additional presence of the next GLB1 processing intermediate GLB1. By immuno-electron microscopy it was possible to localize GLB in globular deposits in PSV in scutellum cells of these somatic embryos.     

Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

Morphology and Anatomy of Pisum Sativum Somatic Embryos

The morphological and anatomical aspects of direct and indirect somatic embryogenesis in pea were described. Direct embryos were induced from shoot apical meristems of 3 to 5-d-old pea seedlings, embryogenic callus originated from immature pea zygotic embryos or shoot apices. Auxin (picloram, 2,4-dichlorophenoxyacetic acid) was necessary to induce somatic embryos. The developmental stages typical for pea zygotic embryos were detected. Globular and heartshaped somatic embryos were morphologically similar to their zygotic counterparts; in contrast, torpedo and cotyledonary somatic embryos displayed great morphological variation, which affected mainly cotyledons (size, shape, number). Based on anatomical sections, possible ways of somatic embryo formation and localization of initiation sites within primary explant tissue have been proposed. The multicellular origin of somatic embryos is supposed in both systems of pea somatic embryogenesis under investigation.     

Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos

Immature zygotic embryos of ginseng produced somatic embryos on MS medium without growth regulators. However, in the culture of mature zygotic embryos, excision of the embryo was required for somatic embryo induction. Somatic embryos formed only on excised cotyledons without an embryo axis or on excised embryos without the plumule and radicle of the axis. This observation suggests that the axis tip of the embryo might suppress somatic embryo production although the cotyledon tissues have predetermined embryogenic competency. To clarify the role of the embryo axis on somatic embryo formation, excised plumules or radicles were placed in direct contact with the basal cut-ends of cotyledons. The adhesion of plumules or radicles highly suppressed somatic embryo formation from cotyledon explants. When an agar block containing exudate from excised plumules or radicles was placed in contact with the cut end of the cotyledon, a similar inhibition was observed. These results suggest that embryogenic competence is suppressed by endogenous inhibitors present in the axis tip of the zygotic embryo.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of<Emphasis Type="Italic"> Cryptomeria japonica</Emphasis> D. Don

This report describes the successful plant regeneration via somatic embryogenesis from immature zygotic embryos of Cryptomeria japonica D. Don. For the induction of embryogenic tissue, we determined that the optimal medium contained N⁶-benzyladenine and 2,4-dichlorophenoxyacetic acid. Immature zygotic embryos that were collected at the end of June yielded embryogenic tissue at the highest frequency. Embryogenic tissues that had proliferated in liquid medium included small and loosely packed cells and elongating or elongated cells. We used ten cell lines to determine the optimal medium for the development of somatic embryos. Induced somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots. Gibberellin A₃ in the germination medium had a positive effect on both the elongation of hypocotyls and the survival of seedlings. The frequencies of induction and germination of somatic embryos differed among the cell lines examined. Most of the seedlings grew normally. This system of somatic embryogenesis required 4–5 months for the regeneration of C. japonica plantlets from immature zygotic embryos.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellin A₃Communicated by F. Sato