Direct Somatic Embryogenesis in Roots of Cichorium: Is Callose an Early Marker?

Direct somatic embryogenesis can be obtained from epidermaland cortical cells in roots from in vitro Cichorium plantlets.The first embryogenic cells are seen after six days of culturein darkness, at 35 °C, in a liquid medium supplemented withNAA (1 x 10^–7 M), 6-dimethylallyl-amino-purine (2·5x 10^–6 M), sucrose (0.03 M) and glutamine (1·7x 10^–3 M). Embryogenic cells undergo first a linear andthen a globular segmentation, with increasing cytoplasmic density.These cells and young embryoids show aniline blue fluorescence.SEM allows the same microglobular pattern to be seen on thesurface of young embryoids and on young microspores of Cichoriumused as controls. In this root system, callose deposition seemsto be an early marker in somatic embryogenesis. Somatic embryogenesis, callose, Cichorium     

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     

SEM Characterization of an Extracellular Matrix Around Somatic Proembryos in Roots of Cichorium

Roots of in vitro plantlets of a hybrid Cichorium intybus L.x C. endivia L. placed for 10 d in an agitated liquid inductionmedium in darkness at 35 °C give somatic embryos of varioussizes which disrupt the epidermis. Proembryos can be observedinside the root; they show a fibrillar network linking the surfacecells. The network is observed in agitated and static liquidmedium; it is not well developed on solid medium. It is notremoved by lipid solvents and pectinase but disappears partlywith protease. Ether-methanol (1:1 v/v) for 45 min and Tris-HC1buffer for 3 h at 30 °C destroy it. As in animal cells suchexternal proteic networks are constituents of an extracellularmatrix linked to the cytoskeleton, we tested microtubule destabilizationby colchicine and cold treatments which removed the network;this effect was reversed by DMSO and high temperature. Cichorium, extracellular matrix, extracellular proteins, somatic embryogenesis     

Somatic Embryogenesis and Its Hormonal Regulation in Tissue Cultures of Freesia refracta

Somatic embryogenesis can be induced in tissue cultures of Freesiarefracta either directly from the epidermal cells of explants,or indirectly via intervening callus. These two pathways ofsomatic embryogenesis can be controlled and regulated by varyingthe combinations and levels of exogenous hormones. When younginflorescence segments were cultured in vitro on modified N₄(MN₄) medium supplemented with 2 mg l^–1 indoleacetic acid(IAA) and 3 mg l^–1 6-benzylaminopurine (BAP), some ofthe epidermal cells began to exhibit the features of embryogeniccells. These cells produced embryoids and developed into newplants through direct somatic embryogenesis. If the same explantswere placed on Murashige and Skoog's (MS) medium containing2 mg l^–1 IAA, 05 mg l^–1 BAP and 05 mg l^–1naphthaleneacetic acid (NAA), pale-yellow translucent nodularcalluses appeared on the surface of the explants. When thiskind of callus was transferred to MN6 medium with 2 mg l^–1IAA and 3 mg l^–1 BAP, embryoids formed which further developedinto plantlets. The regenerated plants were morphologicallynormal and possessed the normal diploid chromosome number of2n = 22. A similar result has also been obtained with youngleaf explants of this plant. The early segmentations of embryogeniccells and the development of embryoids were studied using histologicaland scanning electron microscopic techniques, and the resultshave been discussed in association with the ontogeny and originof the embryoids. Freesia refracta Klatt, somatic embryogenesis, plant regeneration, exogenous hormones     

9-kDa acidic and basic nsLTP-like proteins are secreted in the culture-medium conditioned by somatic embryogenesis in Cichorium

Direct somatic embryogenesis was induced in leaf fragments of the Cichorium ‘474’ genotype. Addition of glycerol to the induction medium allowed a relative synchronization of the first division of the embryogenic cells that only occurs after transfer at day 5 to a medium without glycerol. The abundant presence of 9-kDa extracellular proteins in the culture-medium conditioned by somatic embryogenesis is reported here. Such proteins were also secreted when embryogenesis was initiated in root but were never detected when a non-embryogenic genotype was used as control under the same conditions of culture. Among these proteins, one basic and one acidic isoform were separated through cation-exchange chromatography. Both proteins were recognized by an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). In addition, the partial N-terminal amino acid sequence of each isoform showed similarities with nsLTPs of different plant species. The presence of the acidic nsLTP-like protein was concomitant with the obtention of embryogenic cells during the induction step. The basic form was shown to have only accumulated during the expression step when first divisions of embryogenic cells have occurred. These results allowed us to report, for the first time, the secretion of a 9-kDa acidic nsLTP-like protein in the culture-medium conditioned by plant embryogenic cells.     

Early Cellular Events During Direct Somatic Embryogenesis in Cotyledon Explants of Solanum aviculare Forst.

A detailed light and electron microscope study of early cellularevents at the onset of somatic embryogenesis in cotyledon explantsof Solanum aviculare Forst., cultured on MS medium supplementedwith 1 mg l^–1 2,4-dichloro-phenoxyacetic acid (2,4-D)for periods of 0–12 d in darkness, is described. Examinationsof longitudinal sections in a plane offset from the centralveins indicated that the earliest embryogenic events in explantstook place within the first 3–4 d of culture in the parenchymacells associated with the vascular traces closest to the cutbasal ends of cotyledons. Thereafter, parenchyma associatedwith more distal vascular traces became active in an apparentlysequential manner such that, by the second week of culture,progressive stages of embryogenesis could be observed alongthe lengths of cotyledon sections. Despite the fact that epidermalcells and palisade tissues were exposed directly to the 2,4-Dmedium, initiation of embryogenic development was never observedin cells other than those directly associated with vasculartraces. None of the embryogenic events characterized at theultrastructural level were observed in cotyledons cultured onMS medium in the absence of 2,4-D with the exception that starchaccumulated in decreasing amounts from the wounded basal endto the distal end of each cotyledon. This system provides a valuable model with which to study earlybiochemical and molecular events occurring in explants duringthe onset of somatic embryogenesis because they occur in a predictablefashion at sequentially situated sites along the explant tissues. Somatic embryogenesis, Solanum aviculare, cotyledon explants, cellular changes     

Direct Somatic Embryoid Formation on Immature Embryos of Trifolium repens, T. pratense and Medicago sativa, and Rapid Clonal Propagation of T. repens

By direct somatic embryogenesis in vitro a clone of asepticplantlets can be raised from a single immature embryo of Trifoliumrepens (white clover) within about 6 weeks of pollination. Embryoidsare induced directly from intact zygotic embryonic tissue ona culture medium containing 0·025 or 0·05 mg 1^–1BAP and 1·0 g 1^–1 yeast extract. Similar directsomatic embryogenesis has also been achieved for Trifolium pratense(red clover) and Medicago sativa (lucerne). Applications ofembryo propagation by direct somatic embryogenesis are discussed,particularly in relation to multiple screening of host genotypesfor analysis of host/pathogen and legume/Rhizobium interactions. Trifolium repens L., Trifolium pratense L., Medicago sativa L., clover, lucerne, tissue culture, embryoid, somatic embryogenesis, legumes     

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     

Somatic Embryogenesis and Plant Regeneration from Leaf Callus of Hordeum vulgare

Explants obtained from the basal portion of leaves of Hordeumvulgare (cv. Karan 92) gave rise to callus when cultured onMurashige and Skoog (MS) basal medium supplemented with 2, 4-dichlorophenoxyaceticacid (2, 4-D). Initially, the callus was friable, shiny-whiteand watery but subsequently some compact, nodular callus appeared.The latter were cultured on MS medium containing 0.05 mg l^–12, 4-D and 0.1 mg l^–1 N6-furfurylaminopurine (kinetin),when plantlets were generated. Histological studies showed thatplantlet regeneration occurred by the formation of somatic embryos.The regenerated plants had the normal diploid chromosome number(2n = 14). Hordeum vulgare, barley, somatic embryogenesis, tissue culture, plant regeneration     

Ultrastructural Changes in Cotyledons of Pineapple Guava (Myrtaceae) During Somatic Embryogenesis

Ultrastructural studies (SEM and TEM) were performed on cotyledonsof pineapple guava ( Feijoa sellowiana Berg, Myrtaceae) inducedto form embryos on medium containing 1.0 mg l^-1(4.5µM2,4-D) and 0.3M sucrose. At the time of culture, the cells werefilled with protein and lipid bodies. Microbodies and poorlydifferentiated organelles could also be seen. In contrast togerminating cotyledons, where lipid and protein reserves werequickly metabolized, cells of the embryogenically induced cotyledonsshowed evidence of reserve consumption only after 5 d of culture.Subepidermal cells of the upper cotyledonary surface underwentseveral divisions giving rise to a meristematic layer of severalcells thickness from which somatic embryos developed. Embryoscould also be formed directly by successive divisions of epidermalcells. Cells involved in somatic embryo formation containeda large nucleus with a conspicuous nucleolus and dense cytoplasmwhere numerous ribosomes, mitochondria, plastids with starchand short profiles of rough endoplasmic reticulum were present.Plasmodesmata were present both in cell walls of the meristematiccells and in few celled embryos whereas in degenerating embryosor in more advanced stages of somatic embryo development noplasmodesmata could be found. Although oil bodies were not observedin the meristematic cells they were identified in very youngembryos, being the first reserve compounds to appear. Cellsnot involved in somatic embryo differentiation were characterizedby the presence of several microbodies containing a crystalloidinclusion and elongated mitochondria. Feijoa sellowiana ; pineapple guava; somatic embryogenesis; ultrastructural studies     

Regeneration of Lasiurus scindicus from Tissue Culture

Embryogenic callus cultures were initiated from mature embryosof Lasiurus scindicus on Murashige and Skoog's medium supplementedwith 6 mg l^–1 2,4-Dichlorophenoxyacetic acid (2,4-D).These cultures were maintained on 2 mg l^–1 2,4-D. Plantletswere regenerated via somatic embryogenesis when the calli weretransferred onto hormone-free MS basal medium. Young plantswere successfully transplanted to pots and grown to maturityin a greenhouse. Grass, Lasiurus scindicus, Thar Desert, drought tolerant, somatic embryogenesis, plant regeneration     

Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells

A calmodulin like domain protein kinase (CPK) homologue wasidentified in alfalfa and termed MsCPK3. The full-length sequenceof cDNA encoded a 535 amino acid polypeptide with a molecularweight of 60.2 kDa. The deduced amino acid sequence showed allthe conserved motifs that define other members of this kinasefamily, such as serine-threonine kinase domain, a junction regionand four potential Ca²⁺-binding EF sites. The recombinant MsCPK3protein purified from E. coli was activated by Ca²⁺and inhibitedby calmodulin antagonist (W-7) in in vitro phosphorylation assays.The expression of MsCPK3 gene increased in the early phase ofthe 2,4-D induced alfalfa somatic embryogenesis. Heat shockalso activated this gene while kinetin, ABA and NaCl treatmentdid not result in MsCPK3 mRNA accumulation. The data presentedsuggest that the new alfalfa CPK differs in stress responsesfrom the previously described homologues and in its potentialinvolvement in hormone and stress-activated reprogramming ofdevelopmental pathways during somatic embryogenesis. Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.     

Use of calcium to optimize long-term proliferation of friable embryogenic calluses and plant regeneration in Hevea brasiliensis (Mll. Arg.)

In Hevea brasiliensis (Mll. Arg.), increasing the calcium contentof the friable callus maintenance medium from 3 to 9 mM stimulatedregeneration potential through somatic embryogenesis. This stimulationcould be attributed to the homogeneous cytological structureof calluses, which were formed of undifferentiated cells capableof somatic embryogenesis in optimal culture conditions. Thevery marked increase in the active cell population was sufficientto cause a decrease and a stabilization of water and osmoticpotentials of the calluses, whereas their water content increased.The regeneration capacity of calluses cultured on a medium withadditional CaCl₂ was greater in terms of both quantity (numberof somatic embryos produced was increased 2-fold) and quality(germination efficiency trebled). High CaCl₂ concentrations (9 mM CaCl₂) in the embryogenesisinduction medium favoured somatic embryo development when calluseswere maintained 2 months on the same medium. In this case, additionof benzylaminopurine (BAP) and 3,4-dichlorophenoxy- acetic acid(3,4-D) increased the number of embryos produced (243 embryosg^–1 FW callus) and their germination capacity (27%). These culture conditions were used to determine the optimumembryogenesis induction period. The length of the period affectedboth the intensity of embryogenesis (maximum 56–77 d)and somatic embryo quality (maximum 49–70 d). The bestresults were obtained with a 70 d embryogenesis induction period,within which 355 embryos g^–1 FW callus were obtained,with 35% germination. Key words: Calcium, somatic embryogenesis, long-term culture, water status, histology     

Somatic Embryogenesis and Plant Regeneration from Inflorescence Culture of Java Citronella: (Cymbopogon winterianus)

Embryogenic callus was induced from immature inflorescence segmentsof Java citronella (Cymbopogon winterianus) and maintained for2 years on Murashige and Skoog's medium supplemented with 2,4-D(l mg l^–1). The callus cells retained the original chromosomenumber of 2n = 20. The somatic embryos germinated into plantletson MS basal medium or medium with IAA, NAA, BAP or KN individually(l mg l^–1). The regenerated plantlets developed a goodroot system on full strength solid MS inorganics medium withIAA (1 mg l^–1). The regenerated plants were similar tothe donor plant in morphology and had the same chromosome number,but showed some variation in the essential oil content. Java citronella, Cymbopogon winterianus, somatic embryogenesis, regeneration, inflorescence culture     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture     

SHORT COMMUNICATION Cyclic Nucleotides inFrankiaand Symbiotic Nodules

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine3',5'-monophosphate (cGMP) contents of cultured cells ofFrankiastrainsoriginally isolated from nodules ofAlnus sieboldiana, MyricarubraandElaeagnus macrophyllawere measured by enzyme immunoassays(EIA).Frankiacells, cultured for 59–121 d, had cAMP contentsranging from 2.9 to 76.1 pmol mg^-1protein and cGMP contentsranging from 0.9 to 5.2 pmol mg^-1protein. FollowingFrankiaculture,the media contained extremely large quantities of cAMP and significantlevels of cGMP. The nature of accumulation and secretion ofcyclic nucleotides by slow-growingFrankiacells was comparableto that by a fast-growing actinomyceteStreptomyces lividansTK24,suggesting that secretion of cAMP byFrankiacells may occur throughthe cell membrane but not by cell lysis. cAMP and cGMP contentsin the symbiotic nodules, leaves and roots of actinorrhizalplants and leaves of non-actinorrhizal trees were also measured.The nodules of actinorrhizal woody plants(A. sieboldiana, E.macrophylla, E. umbellata, E. pungensandM. rubra)had cAMP contentsranging from 4 to 258 pmol g^-1f. wt and cGMP contents rangingfrom 1.1 to 5.2 pmol mg^-1protein. Most leaves and some rootsof actinorrhizal plants and all the leaves of non-actinorrhizalwoody plants examined contained small but significant amountsof cAMP and cGMP. This is the first report of significant contentsof cAMP and cGMP in culturedFrankiacells andFrankia-infectednodules. Possible roles of cyclic nucleotides as symbiotic signalsare discussed.Copyright 1998 Annals of Botany Company cAMP, cGMP, actinorrhizal plants, nodules,Frankia,symbiosis.     

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     

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