Histodifferentiation of somatic embryos in cotyledons of pineapple guava (Feijoa sellowiana Berg)

Summary Somatic embryos of pineapple guava (Feijoa sellowiana Berg, Myrtaceae) were induced particularly well from the adaxial face of the cotyledons of zygotic embryos cultured on MS medium containing 1.0 mg/l 2,4-D and 0.3 M sucrose. Somatic embryos were never obtained from globular and heart-shaped zygotic embryos and embryos at the torpedo stage produced somatic embryos at lower frequencies than mature zygotic embryos. At the time of explantation, cotyledonary cells were rich in storage proteins and lipids but no starch was found. After the first 5 days of culture most of the reserves had been mobilized in cotyledons of germinating embryos, but were still present in large amounts in cotyledons undergoing embryogenie induction. In contrast to cotyledons following the normal pattern of development, cells of embryogenically-induced cotyledons accumulated starch, especially those cells not involved in the embryogenie process. Two patterns of somatic embryo differentiation were observed: (1) from single epidermal cells or (2) from groups of meristematic cells near the adaxial surface. Comparative observations on cotyledons from germinating embryos and those undergoing embryogenesis suggest that the meristematic layer arises as the result of successive divisions of cells that, under normal conditions, would form the palisade parenchyma. These were the only mesophyll cells that showed mitotic divisions during the normal development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - FAA formalin/acetic acid/ethyl alcohol - PAS periodic acid-Schiff     

Morphohistological analysis and histochemistry of Feijoa sellowiana somatic embryogenesis

Summary. Morphohistological analysis and histochemical studies were carried out during the induction and development of Feijoa sellowiana somatic embryos. Zygotic embryos were cultured on LPm medium containing 2,4-dichlorophenoxyacetic acid (20µM) and glutamine (8mM). Somatic embryogenesis could be induced from embryogenic cells that originated in meristematic centers or from clusters of cells. The presence of few starch grains and abundant protein bodies was observed in the globular and early torpedo stages, while in torpedo and cotyledonary-stage somatic embryos an enhanced synthesis of starch grains was associated with the accumulation of reserves to be used in the conversion of the embryos to plantlets. Proteins were predominantly observed in protoderm cells, as well as in the meristematic apical region of torpedo and cotyledonary-stage somatic embryos.Correspondence and reprints: Departamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, C.P. 476, 88034-001 Florianópolis, SC, Brazil.     

Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae)

The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.     

Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l⁻¹ α-naphthalene acetic acid and later transferred to MS without growth regulators (MS_WH). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Somatic Embryogenesis in Cassava: The Anatomy and Morphology of the Regeneration Process

Anatomical and morphological studies demonstrated that somaticembryos developed similarly on mature seed and clonal leaf explantsof cassava (Manihot esculenta Crantz) cultured for 20–24d on Murashige and Skoog (MS2) basal medium supplemented with4.0 mg l^–1 2,4-D (Stage 1) before transfer to MS2 basalmedium supplemented with 0–01 mg l^–1 2,4-D and 0–1mg l^–1 6-benzylaminopurine (Stage II medium). Within 7d of inoculation onto Stage I medium, cell divisions occurredin the adaxial tissues of cotyledon-piece and leaf-lobe explants,and associated with this was the development of embryogeneticprotusions and ridges on the adaxial surface. Foliose structuresand somatic embryo initials developed from these tissues oncotyledon, embryonic axis and leaf-lobe explants and, when cultureswere transferred to Stage II medium, further somatic embryodevelopment occurred. Somatic embryos apparently originatedfrom groups of cells and were identified by the presence ofa closed root axis, a shoot axis and cotyledons of similar shapeand venation to those of zygotic embryos. Somatic embryos hadno vascular connection with parental cultures. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, anatomy, morphology, morphogenesis     

Ultrastructural Studies of Somatic Embryos of Eucalyptus nitens and Comparisons with Zygotic Embryos Found in Mature Seeds

Although somatic embryogenesis has been observed in tissuesfrom a limited number of Eucalyptus species cultured in vitro,no comparisons have been made of the morphology and structureof eucalypt somatic embryos and zygotic embryos found in matureseeds. We used scanning and transmission electron microscopy,in conjunction with histological analysis, to compare maturezygotic embryos with somatic embryos of the commercially-importanttemperate eucalypt Eucalyptus nitens. Apart from differencesin the nature of the outer coating enclosing both embryo types,somatic embryos of E. nitens were observed to have strong similaritieswith zygotic embryos in seeds in terms of their overall size,morphology and internal cellular organization. Many cells inboth sexually-produced and somatic embryos contained numerouslipid-rich globular bodies. The wider significance of theseobservations is discussed with regard to their potential applicationsin eucalypt plantation biotechnology programmes. Copyright 2000Annals of Botany Company Eucalyptus nitens, shining gum, somatic embryo, tissue culture, ultrastructure, zygotic embryo     

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.     

Histology of Callogenesis and Somatic Embryogenesis Induced in Stem Fragments of Cork Oak (Quercus suber) Cultured In Vitro

Calluses able to produce somatic embryos were formed duringin vitro culture of shoot fragments of cork oak (Quercus suberL.).Histological monitoring of these fragments during cultureshowed that it was the cortical parenchyma cells which underwentdedifferentiation before calluses were formed by repeated divisions.The calluses consisted of parenchyma cells surrounded by a fewlayers of meristematic cells. Proembryos formed in groups aroundthe edge of some calluses. Histological examination showed thatthey were produced by the evolution of two different categoriesof cell: one category had the appearance of ‘embryogenic’cells with very thick walls, a small vacuole rich in starchand a well-developed nucleus with a prominent nucleolus. Theother cells were very bulky with large vacuoles; their morphologywas similar to that of suspensor cells encountered in embryogenesisin gymnosperms. The ontogenic stages were similar to those describedin zygotic embryos of the genus Quercus. Nevertheless, mostof the embryonic structures deviated from normal developmentand at all stages produced secondary proembryos. Cork-oak, Quercus suber L, histology, callogenesis, somatic embryogenesis, embryogenic cells, starch, secondary embryogenesis     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

Secretion of Specific Extracellular Proteins by Somatic Embryos of Picea abies is Dependent on Embryo Morphology

Embryogenic cell lines ofPicea abieswere categorized into twogroups, A and B, based on the morphology of the somatic embryosand the ability of the somatic embryos to proceed through amaturation process when treated with ABA. Group A embryos hada distinct, densely-packed embryonic region whereas group Bembryos had loosely packed cells in their embryonic region.Embryo morphology was shown to be regulated by changes in theplant growth regulators in the culture medium. Treatment withN⁶-benzyladenine stimulated embryos to develop large embryonicregions. The morphology of somatic embryos and especially thatof the embryonic regions was correlated with the presence ofspecific extracellular proteins. Only somatic embryos with denselypacked cells in the embryonic regions secreted proteins withrelative molecular weights of 28, 66 and 85kD. The extracellularprotein of 28kD was isolated and the first 21 amino acids inthe N-terminus were identified. These showed 52–57% identitywith the N-terminal sequence conserved among members of a proteinfamily which includes zeamatin and which have been shown tobe involved in plant anti-fungal mechanisms. Immunological studiesof extracellular chitinases and zeamatin-like proteins, as wellas of activity of extracellular peroxidase, revealed a closecorrelation between the presence of specific chitinases andembryo morphology. Auxin; cytokinin; embryogenic cell lines; embryo morphology; extracellular proteins; Norway spruce; Picea abies; somatic embryos     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

There is No Somatic Meiosis in Embryogenic Leaves of Cichorium

Several papers dealing with carrot cell cultures describe meiosis-likedivisions and haploid cells prior to somatic embryogenesis.We have studied the first division in embryogenic mesophyllcells of a diploidCichorium intybus L. and of a tetraploid hybridC.intybus L.xC. endivia L. which undergo direct somatic embryogenesisfrom single cells when leaf fragments are placed in a liquidagitated inductive medium (modified MS with 1x10^-7M NAA and2.5x10^-6M 2-iP), in darkness, at 35°C. MicrosporogenesisinC. intybus provided aspects of meiosis for comparison. Inleaves incubated in inductive conditions, DAPI staining of nucleishowed normal mitosis on days 3–6; about 0.6% cells inprophase had undergone spontaneous endoreduplication leadingto a tetraploid somatic embryo. Immunocytochemistry of tubulinrevealed the constant presence of a preprophase band, as ina normal mitosis. The first pluricellular somatic embryos becamevisible on day 5 of culture. Flow cytometric determination ofnuclear DNA on days 4, 5 and 6 did not show any peak correspondingto the 1C DNA level for the diploid plant or to the 2C DNA levelfor the tetraploid. Instead there was a weak but constant peakat the 4C and 8C levels. We conclude that inCichorium leaves,the first division of somatic embryogenesis is a normal mitosis,with a small shift to endoreduplication. In our opinion, somaticmeiosis is not a prerequisite during direct somatic embryogenesis. Cichorium ; chicory; somatic embryogenesis; cell division; flow cytometry; tubulin     

Ontogenesis, Differentiation and Precocious Germination in Anther-derived Somatic Embryos of Grapevine (Vitis vinifera L.): Proembryogenesis

Histological steps of callogenesis and proembryogenesis in anthercultures ofVitis vinifera L. ‘Grenache noir’ aredescribed. Embryogenic calli were obtained on Nitsch and Nitschmedium supplemented with 1mgl^-12,4-dichlorophenoxyacetic acid(2,4-D) and 0.25mgl^-1benzylaminopurine (BAP). Calli were initiatedfrom anther connective cells only and no division of microsporesoccurred. The embryos were hence of somatic origin. Proembryosdeveloped either directly (i.e. without intervening callus)from the endothecium, or indirectly from the connective-derivedcallus. In both cases, proembryos originated from single cells.They developed from starchy differentiated cells of a predeterminedtype. The polarity of the somatic proembryo was establishedfrom the first divisions and it was marked by precocious developmentof an easily recognizable suspensor. Other analogies with thedevelopment of the zygote are also emphasised. Vitis vinifera L.; grapevine; somatic embryogenesis; proembryogenesis; histology     

Ontogeny of Somatic Embryos of Vigna aconitifolia, Vigna mungo and Vigna radiata

Callus cultures were initiated from immature cotyledons of Vignaaconitifolia, V. mungo and V. radiata on MS medium supplementedwith NAA, picloram or 2, 4-D. On transfer to L-6 liquid mediumsupplemented with low concentrations of picloram, GA₃ and cytokinins,large number of somatic embryos differentiated from the callus.The cells destined to become somatic embryos divided to formspherical or filamentous proembryos. From the filamentous proembryo,the embryo proper developed either at single or multiple sites.Development of somatic embryos from multiple sites resultedin several embryos connected by a common suspensor at the radicleend. Continued divisions of the proembryos led to globular,heart shaped and dicotyledonary stages of somatic embryogenesis.The somatic embryos of V. mungo and V. aconitifolia differentiatedinto tiny plantlets at low frequency (1%) in liquid suspensioncultures supplemented with zeatin, picloram and GA₃. Vigna aconitifolia Jacq, Marechal, mothbean, Vigna mungo L. Hepper, urdbean, Vigna radiata L. Wilczk, mungbean, somatic embryo     

The ultrastructure of somatic embryo development in pearl millet (Pennisetum glaucum; Poaceae)

The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.     

High Frequency of Plant Production via Somatic Embryogenesis from Callus or Cell Suspension Cultures inEleutherococcus senticosus

Hypocotyl segments ofEleutherococcus senticosuscultured on Murashigeand Skoog's (MS) medium with 4.5 µM2,4-D produced somaticembryos directly from the surface of explants without interveningcallus formation. When these somatic embryos were subculturedto the same MS medium with 4.5 µM2,4-D, friable embryogeniccalli were formed mainly from radicle tips of somatic embryos,but at a low frequency (5%). Selected embryogenic calli weremaintained on MS agar or liquid medium with 4.5 µM2,4-D.To induce somatic embryo development, embryogenic calli andcell clumps were transferred to MS medium lacking 2,4-D. Thefrequency of somatic embryo formation differed between culturetypes with 1570 embryos formed per Petri dish from callus cultureand 5514 embryos formed per flask from cell suspension cultures.Somatic embryos formed on agar medium had larger cotyledonsthan those of embryos formed in liquid medium. GA₃treatmentwas necessary to induce germination from somatic embryos. Therate of plant conversion was 97% in somatic embryos from callusculture and 76% in embryos from liquid culture. Regeneratedplantlets were successfully acclimatized in the glasshouse.Copyright1999 Annals of Botany Company Eleutherococcus senticosus, micro propagation, somatic embryogenesis.     

Effect of NaCl Salinity on Somatic Embryo Development in Sapindus trifoliatus L.

The effect of NaCl salinity on growth and development of somaticembryos of Sapindus trifoliatus L. was examined. Incorporationof 25 and 50 mol m^–3 NaCl into the medium greatly increasedthe growth and development of somatic embryos and both theseconcentrations favoured the production of secondary embryoids.However, supplementation of 100 mol m^–3 NaCl to the mediumdid not have any significant effect on the growth and developmentof somatic embryos. On the other hand, the culturing of proembryostructures in medium containing 200 mol m^–3 NaCl resultedin complete death within 7 d of salt exposure. Analysis of somatic embryos revealed that, upon salinization,they accumulated Na⁺ and Cl^– in significant amounts butthe content of Na⁺ was much less compared to that of Cl^–.Addition of NaCl (up to 50 mol m^–3) in the medium resultedin a considerable increase in the K⁺ content of somatic embryos.The content of proline in somatic embryos, however, increasedsubstantially in response to salinization. The amount of freesterols, steryl glycosides, steryl esters, and phospholipidsalso rose to higher values in salt-affected somatic embryos.The results suggest that somatic embryos of S. trifoliatus cantolerate concentrations of NaCl up to 100 mol m^–3 withoutaffecting growth and that they have sufficient cellular mechanismsto tolerate salinity at relatively high levels. Key words: Salinity, somatic embryo, sterols, phospholipids     

Anatomical Sequence and Morphometric Analysis during Somatic Embryogenesis on Cultured Cotyledon Explants of Camellia japonica L.

This article describes the origin and anatomical developmentof somatic embryos differentiated on Camellia japonica L. cotyledonscultured on Murashige and Skoog's medium containing 1 mg l^-1of 6-benzylaminopurine. Only the abaxial surface of the cotyledonexplants was morphogenetically competent. Embryos developedin abaxial parenchymatic protuberances or nodules arising bydedifferentiation and active cell division in the epidermisand subepidermis. After 12-15 d in culture, successive divisionsat the surface of the nodules led to the formation of embryogenicprecursor cells which dedifferentiated into embryogenic cells;most somatic embryos apparently had a multicellular origin frommulticellular proembryonal complexes, though a number of few-celledproembryos within a thick common wall seemed to have originatedunicellularly. Between days 24 and 27, somatic embryos at theheart-shaped, torpedo-shaped and cotyledonary stages were apparent.Computer-aided image analysis of the histological events showeda progressive increase in the nucleus-to-cell area ratio. Duringthe first 7 d culture the explants exhibited a rapid declinein protein body content, which was high in the initial cotyledon,and an increase in starch content. Developing nodules stronglyPAS-positive, but starch content subsequently declined in thetissues underlaying embryogenic areas and reached a minimumwhen somatic embryos developed.Copyright 1993, 1999 AcademicPress Camellia japonica, camellia, cellular changes, somatic embryogenesis, histology, image analysis