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     

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     

Origin and Development of Somatic Embryoids Formed Directly on Immature Embryos of Trifolium repens In Vitro

When immature zygotic embryos of Trifolium repens are culturedin vitro in the presence of 0.05 mg 1^–1 BAP, the cellsof the hypocotyl epidermis proliferate to produce somatic embryoidsdirectly without an intervening callus phase. The young epidermalcells show features of proembryogenic cells, and the first signof embyroid induction is a shift from regular equational, anticlinaldivisions to irregular, periclinal and oblique quantal divisions.Multicellular budding and single-cell initiation apparentlyboth occur, with multicellular budding being the more frequentpattern in the present study. Most early proembryoids resembleglobular zygotic proembryos but appear to lack a suspensor.It is suggested that the subtending embryonic tissue fulfilsthe role of a suspensor or proembryonal complex. Secondary proliferationsfrom the young cotyledon and hypocotyl epidermis of primaryembryoids are formed by processes similar to those producingprimary embryoids, and also from structures initially resemblingepidermal hairs. These hair-like structures arise from singlesuperficial cells which show evidence of cutinisation and callosedeposition suggesting some degree of physical separation fromneighbouring cells. Trifolium repens, tissue culture, somatic embryogenesis, embryoid, legume     

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     

Change of Superoxide Dismutase Activities During Somatic Embryogenesis in Asparagus officinalis L.

The superoxide dismutase activities in callus and somatic embryoids at different developmental stages of Asparagus officinalis L. were determined by means light of induced oxidation-reduction of nitro blue tetrazolium. It was shown that the superoxide dismutase activity was higher in callus than in somatic embryoids at different developmental stages. The activity increased with growth, differentiation and maturation of somatic embryoids during somatic embryogenesis. The relations between superoxide dismutade activity and somatic embryogenesis were discussed.     

A novel method for increasing the frequency of somatic embryogenesis in wheat tissue culture by NaCl and KCl supplementation

The effect of NaCl, KCl and LiCl on the growth and morphogeneis of tissue cultures originating from immature embryos of four wheat (Triticum aestivum L.) and one triticale (Triticosecale)varieties was investigated. The morphogenetic pathway to plant regeneration in Chinese Spring wheat was determined as incomplete somatic embryogenesis because the differentiation and subsequent germination of the shoot apices happened in the early phase of embryo development. Culture medium supplemented by NaCl suppressed the differentiation of shoot apices resulting in the development of more typical somatic embryoids. Forty mM concentrations of both NaCl or KCl increased the formation of somatic embryos in Chinese Spring. Arthur and GK Kincso wheat varieties while Lasko triticale regenerated well without the addition. The salts inhibited plantlet formation from somatic embryoids so the salts supplement should be omitted. Forty mM LiCl inhibited growth while 10mM LiCl had no effect on growth or embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Development of Pollen Embryoids in Anther Cultures of Datura innoxia: I. GENERAL OBSERVATIONS AND EFFECTS OF PHYSICAL FACTORS

To obtain the maximal production of pollen embryoids in culturedanthers of Datura innoxia, the critical stage of anther developmentand the effect of physical factors, such as the precise modeof implantation of the anthers in the culture medium, light,temperature, and pH, were studied. In almost all media used,anthers containing uninucleate pollen were the best for initiationof embryogenesis. Variations in light and temperature also affecteddevelopment of the embryoids significantly. The percentage ofanthers producing pollen embryoids increased almost linearlywhen the temperature was raised from 22 to 30 °C. At lowertemperatures (15 to 20 °C) no embryoids were produced. Cultureskept in darkness produced embryoids, but upon transfer of culturesto the light the percentage of responding anthers increasedconsiderably.     

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     

金花茶子叶在离体培养中胚状体的发生和小植株的形成

研究了金花茶(Camellia chrysantha(Hu)Tuyama)子叶在离体培养中体细胞胚状体发生的条件。在MS基本培养基中附加苄基嘌呤(BA)或苄基嘌呤与萘乙酸(NAA)组合,诱导了胚状体发生。组织学观察表明,胚状体起源于子叶的表皮细胞。在增添细胞分裂素和生长素的MS或改良B_5液体培养基里振荡培养,明显地促进了胚状体根的生长和茎的发育。胚状体在继代培养中能保持旺盛的再生能力。已得到两个繁殖率较高的胚状体无性系。在合适的条件下,胚状体能长成正常的小植株。     

Control of Embryoid Development in Tissue Cultures of Celery

Scanning electron microscope photographs of the embryoids showedglobular embryoids attached to the surface of aggregates inliquid medium and also some free floating. The surface structureof the unattached embryoids was very irregular, but, with thechange to polarized growth in the heart and torpedo forms, thesurface of the embryoid became smoother. The stage of developmentof the embryoids could be controlled by modifying the compositionof the medium to the extent that the majority of the embryoidsin the culture were either globular or torpedo forms. One ofthe most effective compounds in controlling development was2,4–dichlorophenoxy acetic acid (2,4–D). At high2,4–D concentrations, embryogenesis in the callus wasrestricted to the globular stage and after two subcultures itwas totally repressed, while after ten subcultures the potentialfor embryogenesis was lost and could not be regained even aftersubculture on a normal medium. On the normal agar medium thecallus always continued to show embryogenesis, but when it wastransferred to liquid medium of the same composition, embryoidswere produced in the first subculture but the potential haddeclined by the third subculture, when only roots were produced,and after ten subcultures cell growth and all differentiationwas totally it hibited. However, in the first subculture inliquid medium, embryogenesis was sequential with the whole cultureprogressing from globular to torpedo forms. This was particularlyeffective when the callus inoculum had been maintained on ahigh 2,4–D concentration for the two subcultures priorto inoculation of the liquid medium. By making use of this sequentialchange in embryoid development, a large number of embryoidscould be obtained at any particular stage. Apium graveolens, celery, tissue culture, embryoids, 2,4–D     

Histological Analysis of Organogenesis and Somatic Embryogenesis Induced in Immature Tissues of Stylosanthes scabra

A well established protocol for in vitro germination of Stylosanthesscabra zygotic embryos was achieved. The response of S. scabraembryonic tissues cultured in vitro was highly dependent onthe kind of growth regulator used. Organogenesis was obtainedby using BAP, otherwise somatic embryogenesis was induced by2, 4-D. Histological aspects of both methods of regenerationwere evaluated. Endogenous neoformed buds seem to develop fromdeepseated vascular nodule structures into callus tissue. Besides,a direct somatic embryogenesis of a multicellular origin issuggested. Stylosanthes scabra, histology, organogenesis, somatic embryogenesis     

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     

Somatic Embryogenesis and Regeneration of in Plantlets in Pomegranate

Plantlets were regenerated from somatic embryos originatingfrom cotyledonary tissues of pomegranate (Punica granatum) throughmultiple somatic embryogenesis. Embryogenic cell clusters proliferatedvigorously with regular sub-culturing after 20 d on RBM-II mediumcontaining 1 µM kinetin (KN), 2 µM benzylamino purine(BAP) and 5 µM 2,4-dichlorophenoxyacetic acid (2, 4-D).Developmental stages of somatic embryos were expressed on sub-culturingwith a low level of 2, 4-D (2.5 µM). Embryogenic initialscells were small, round to oval, thick-walled, contained densecytoplasm which stained with acetocarmine and were usually attachedto non-embryogenic cells. Embryo maturation was obtained onRBM-III and IV media to produce young seedlings on the initiationof the first long tap root. Punica granatum L., pomegranate, multiple somatic embryogenesis, callus culture, plant regeneration     

DIRECT EMBRYOGENESIS FROM CULTURED ANTHERS AND PISTILS OF DACTYLIS GLOMERATA

Direct somatic embryoids were initiated from orchardgrass (Dactylis glomerata L.) anthers and unpollinated pistils cultured in the dark at 25 C on Schenk and Hildebrandt (SH) medium supplemented with 30 μM dicamba (3,6 dichloro-o-anisic acid). Stereoscopic and scanning electron microscopy indicated that the embryoids originated from anther walls and from ovary and style regions of pistils. Callus initiation from direct embryoids leading to secondary embryogenesis was observed in pistils cultured from 4–6 wk. The ability of these calli to proliferate and initiate new embryoids through the dedifferentiation and redifferentiation of preexisting embryoids suggests long-term totipotency.     

Morphohistological and flow cytometric analyses of somatic embryogenesis in <Emphasis Type="Italic">Trifolium nigrescens</Emphasis> Viv.

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l⁻¹ N⁶-[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygotic-like structures to abnormal structures including horn-shaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets.     

小麦幼胚培养中的体细胞胚胎发生

小麦品种崇阳红麦和鄂思一号杂种一代幼胚培养具有再生植株的潜力。从一个幼胚经200天左右的连续培养获得530多株再生植株,并从中获得了典型的具有两极性的与愈伤组织块仅局部相连的胚状体。体细胞胚胎发生是小麦幼胚培养的主要途径,但受培养条件的影响,以MS培养基作基本培养基,低浓度2,4-D(0.4mg/1)和水解酪蛋白(1000mg/l)有利于体细胞胚胎发生。     

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.     

Spatial distribution of free and conjugated polyamines in leaves of Solanum melongena L. associated with differential morphogenetic capacity: efficient somatic embryogenesis with putrescine

In eggplant (Solanum melongena L., cv. Pusa Purple Long), explantsfrom different regions of the leaf showed significant differencesfor embryogenic potential. Discs from the apical region of leavesyielded more somatic embryos than those from the basal region.Apical discs showed consistently higher polya-mine titres thanthe basal discs. Putrescine treatment promoted somatic embryogenesisand at 0.5 mM it caused a remarkable increase (c. 6-fold) ina number of somatic embryos, accompanied by an increased putrescinetitre. On the other hand, spermidine and spermine had no stimulatoryeffect on embryogenesis; rather they were inhibitory at higherconcentrations. All tested inhibitors of polyamine biosynthesissuch as difluoromethylarginine, difluoromethylomithine, methylglyoxalbis (guanylhydrazone) and bis (cyclohex-ylammonium) sulphatesignificantly inhibited somatic embryogenesis. Difluoromethylarginineblocked somatic embryogenesis by lowering endogenous polyaminecontents (particularly putrescine) and such inhibitory effectswere totally restored by exogenous putrescine (0.5 mM), concomitantwith the revival of endogenous PA concentrations. These resultsdemonstrate (i) a positive correlation between the spatial distributionof free and conjugated polyamines and the embryogenic capacityof an explant and (ii) putrescine caused the promotion of somaticembryogenesis, suggesting the intricate regulatory role of polyamines,specifically putrescine, in somatic embryogenesis in eggplant. Key words: Solanum melongena, somatic embryogenesis, position effect, polyamines, putrescine, polyamine biosynthesis inhibitors, difluoromethylarginine     

REVIEW ARTICLE: Zygotic embryogenesis versus somatic embryogenesis

This review will summarize molecular and genetic analyses aimedat identifying the mechanisms underlying the sequence of eventsduring plant zygotic embryogenesis. These events are being studiedin parallel with the histotogical and morphological analysesof somatic embryogenesis. The strength and limitations of somaticembryogenesis as a model system will be discussed briefly. Theformation of the zygotic embryo has been described in some detail,but the molecular mechanisms controlling the differentiationof the various cell types are not understood. In recent yearsplant molecular and genetic studies have led to the identificationand characterization of genes controlling the establishmentof polarity, tissue differentiation and elaboration of patternsduring embryo development. An investigation of the developmentalbasis of a number of mutant phenotypes has enabled the identificationof gene activities promoting (1) asymmetric cell division andpolarization leading to heterogeneous partitioning of the cytoplasmicdeterminants necessary for the initiation of embryogenesis (e.g.GNOM),(2) the determination of the apical-basal organization whichis established independently of the differentiation of the tissuesof the radial pattern elements (e.g.KNOLLE, FACKEL, ZWILLE),(3) the differentiation of meristems (e.g.SHOOT-MERISTEMLESS),and (4) the formation of a mature embryo characterized by theaccumulation of LEA and storage proteins. The accumulation ofthese two types of proteins is controlled by ABA-dependent regulatorymechanisms as shown using both ABA-deficient and ABA-insensitivemutants (e.g.ABA, ABI3). Both types of embryogenesis have beenstudied by different techniques and common features have beenidentified between them. In spite of the relative difficultyof identifying the original cells involved in the developmentalprocesses of somatic embryogenesis, common regulatory mechanismsare probably involved in the first stages up to the globularform. Signal molecules, such as growth regulators, have beenshown to play a role during development of both types of embryos.The most promising method for identifying regulatory mechanismsresponsible for the key events of embryogenesis willcome frommolecular and genetic analyses. The mutations already identifiedwill shed light on the nature of the genes that affect developmentalprocesses as well as elucidating the role of the various regulatorygenes that control plant embryogenesis. Key words: Development, marker, mutant, somatic embryogenesis, zygotic embryogenesis     

Somatic Embryogenesis: Factors Influencing Coordinated Behaviour of Cells as an Embryogenic Group

A number of common features are associated with a great diversityof observations of somatic embryogenesis in vitro. There arefundamental homologies between direct and indirect somatic embryogenesis,and between single-cell and multiple-cell initiation. Many ofthe observed differences can be attributed to whether or notcells require redetermination to the embryogenic state, andto differences in the nearest neighbour relationships of initiatingcells. The observed pattern of morphogenesis depends on whethera group of cells can establish and maintain coordinated behaviouras an embryogenic unit and will be influenced by factors whichaffect intercellular communication. Somatic embryogenesis, tissue culture, cell-cell interactions