DEVELOPMENTAL ANATOMY OF DIRECT SHOOT ORGANOGENESIS FROM LEAF PETIOLES OF VITIS VINIFERA (VITACEAE)

The anatomy of direct shoot organogenesis from leaf petioles of Vitis vinifera cv. French Colombard cultured in vitro was studied by light microscopy. Regenerating petiole stubs were fixed at 2- or 3-day intervals and sectioned longitudinally. By day 3 on regeneration medium, new cell divisions were observed. After 6 days, three distinct regions of meristematic activity were apparent within the expanding petiole stub: the wound-response, organogenic, and vascularization regions. In the organogenic region, rapid periclinal divisions of vacuolate outer cortical cells formed nodular bumps, many of which developed vascular strands and marginal meristems and formed adventitious leaves. Promeristems with small, densely staining cells and a distinct tunica layer also originated in the organogenic region, by cell division in the epidermal and subepidermal cell layers. With vascularization and the formation of leaf primordia, many promeristems became adventitious shoot meristems. Adventitious leaves and promeristems were initiated continuously from day 10 until day 33. Promeristems were often initiated near or upon adventitious leaves but could form either before or after the adventitious leaf developed. Adventitious leaves and shoot meristems developed vascular connections with the vascular bundles of the original expiant. The implication of this pattern of regeneration for Agrobacterium-mediated transformation of Vitis is discussed.     

Morphohistological analysis of the origin and development of somatic embryos from leaves of mature Quercus robur

Summary In oak species, there is paucity of information on the anatomical changes underlying differentiation of somatic embryos from explants of mature trees. A histological study was undertaken to ascertain the cellular origin and ontogenesis of somatic embryos in leaf cultures from a 100-yr-old Quercus robur tree. Somatic embryogenesis was induced in expanding leaves excised from shoots forced from branch segments, following culture on three successive media containing different concentrations of α-naphthaleneacetic acid and 6-benzylaminopurine. The somatic embryogenesis followed an indirect pathway from a callus tissue formed in the leaf lamina. After 4–6 wk of culture, meristematic cells originated in superficial layers of callus protuberances, but these cells evolved into differentiated vacuolated cells rather than embryos. A subsequent dedifferentiation into embryogenic cells occurred later (9–12 wk of culture) within a dissociating callus. Embryogenic cells exhibited dense protein-rich protoplasm, high nucleoplasmic ratio, and contained small starch grains. Successive divisions of these cells led to the formation of a few-celled proembryos and embryogenic cell clumps within a thick common cell wall, which seemed to have originated unicellularly. However, a multicellular origin of larger embryogenic clumps could not be dismissed; these gave rise to embryonic nodular structures that developed somatic embryos of both uni- and multicellular origin. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were apparent.     

SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root

The primary root of Arabidopsis has a simple cellular organisation. The fixed radial cell pattern results from stereotypical cell divisions that occur in the meristem. Here we describe the characterisation of schizoriza (scz), a mutant with defective radial patterning. In scz mutants, the subepidermal layer (ground tissue) develops root hairs. Root hairs normally only form on epidermal cells of wild-type plants. Moreover, extra periclinal divisions (new wall parallel to surface of the root) occur in the scz root resulting in the formation of supernumerary layers in the ground tissue. Both scarecrow (scr) and short root (shr) suppress the extra periclinal divisions characteristic of scz mutant roots. This results in the formation of a single layered ground tissue in the double mutants. Cells of this layer develop root hairs, indicating that mis-specification of the ground tissue in scz mutants is uncoupled to the cell division defect. This suggests that during the development of the ground tissue SCZ has two distinct roles: (1) it acts as a suppressor of epidermal fate in the ground tissue, and (2) it is required to repress periclinal divisions in the meristem. It may act in the same pathway as SCR and SHR.     

Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L.

Summary Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

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     

Histology of Shoot Bud Ontogeny from Seedling Root Segments of Brassica napus L.

Root explants of Brassica napus cultured in vitro form adventitiousshoots. The root buds originated at the base of the newly initiatedlateral root. Cells in association with the differentiatingphloem of the developing lateral roots were the sites for rootbud formation. A nodular mass of cytoplasmic cells developedby day 7 at the base of the lateral root. This group of cellscontinued to divide an enlarge. The cells in the peripheralregion of the nodular cell mass differentiated further intoa meristematic zone. The meristematic cells grew towards theperiphery of the cortex by crushing the outer layer of corticalcells. Further development of the meristematic layer resultedin the formation of shoot primordia with organized shoot apicalmeristems and leaf primordia.Copyright 1993, 1999 Academic Press Brassica napus, canola, cultured root segments, root buds     

Protective effect of wheat germ agglutinin on the course of mitosis in the roots of <Emphasis Type="Italic">Triticum aestivum</Emphasis> seedlings exposed to cadmium

Effect of pretreatment with 28 nM wheat germ agglutinin (WGA) on cell divisions in the root apical meristem of 4-day-old seedlings of wheat (Triticum aestivum L.), distribution of cells among mitotic phases, cadmium-induced disruptions of normal progression through mitosis, and activity of nucleolar organizer regions (NOR) of the chromosomes was studied after 7-h-long exposure to 1 mM cadmium acetate. Pretreatment with WGA has a pronounced protective effect on divisions of root meristem cells exposed to cadmium. Progression of the cells through mitotic phases was normalized, abnormal mitoses became much less numerous, and the share of binuclear cells decreased. Activity of NOR remained at the control level that much depended on the ability of WGA to prevent reduction in cytokinin content under cadmium stress.     

Histological Study of Adventttious bud Formation on Lactuca Sativa Cotyledons Cultured In Vitro

Abstract

Cyto-histological changes accompanying the formation of adventitious buds in excised cotyledons of Lactuca sativa were studied during the first 12 days after planting in vitro. Prospective proliferating cells can first be recognized, already on the first day after planting, by a marked increase in nuclear and nucleolar volumes, followed on the second day by a burst of cell divisions involving particularly mesophyll cells. Then lignified elements develop together with meristematic center, forming a callus-like tissue in the inner part of the cotyledons. At the third day of culture, the epidermal cells start to divide with a periclinal wall followed by an anticlinal division. In the following days of culture the epidermal cells, which divide mainly with periclinal walls, form layers of cells below the surface, gradually filling up the intercellular spaces. From the 8^th day on, the buds protude above the surface and develops into shoots. These results are discussed in relation to DNA content of nuclei of Lactuca sativa cotyledons and to the time course of cell division and tracheary element formation. The very regular sequence of changes associated with the initiation and development of the bud makes the in vitro culture of Lactuca cotyledons an appropriate System for histochemical and biochemical studies.     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.     

Cell lineage analysis of maize bundle sheath and mesophyll cells

Maize leaves are divided into repeated longitudinal units consisting of vascular tissue, bundle sheath (BS), and mesophyll (M) cells. We have carried out a cell lineage analysis of these cell types using six spontaneous striping mutants of maize. We show that certain cell division patterns are preferentially utilized, but not required, to form the characteristic arrangement of cell types. Our data suggest that early in development a central cell layer is formed, most frequently by periclinal divisions in the adaxial subepidermal layer of the leaf primordium. Lateral and intermediate veins are initiated in this central layer, most often by divisions which contribute daughter cells to both the procambium and the ground meristem. These divisions generate "half vein" units which comprise half of the bundle sheath cells around a vein and a single adjacent M cell. We show that intermediate veins are multiclonal both in this transverse direction and along their lengths. BS cells are more closely related to M cells in the middle layer of the leaf than to those in the upper and lower subepidermal layers. An examination of sector boundaries has shown that photosynthetic differentiation in M cells is affected by the phenotype of neighboring BS cells.     

Histology of the Morphogenic Response in Thin Cell Layer Explants from Vegetative Tobacco Plants

The morphogenic response of thin cell layers (TCLs) from vegetativetobacco (Nicotiana tabacum L.) plants can be directed very preciselyby varying the concentrations of benzyladenine (BA) and -naphthaleneacetic acid (NAA) in the culture medium. Medium containing 1·6µM BA and 0·5 µM NAA was optimal for shootformation, concentrations of 0·5 µM BA and 1·6µM NAA were optimal for the induction of shoots and rootson the same explant, whereas concentrations of NAA higher than16 µM resulted in callus proliferation only. Polarityin the distribution of the shoot buds was observed, i.e. a switchfrom basal to apical shoot formation occurred with increasingNAA concentrations, suggesting basipetal transport of NAA. Histologicalexamination of TCLs on shoot induction medium revealed thatfirst cell divisions occurred within 2 d in cortical cells whichwere directly in contact with the medium along the longitudinalcut surface, and after 2 d in subepidermal cells along the lateraledges of the explants. Individual lateral buds originated fromone subepidermal and one or more epidermal cells, while apicalbuds originated from single subepidermal or cortical cells locateddirectly at the apical end of the explant. After culture ofTCLs for 2-3 d on root/shoot induction medium cells in the regeneration-competentsubepidermis elongated, while on callus induction medium subepidermalcells elongated and dedifferentiated. The regeneration systemas described in this study will be used to identify cells competentfor regeneration as well as for transformation.Copyright 1994,1999 Academic Press Nicotiana tabacum L., tobacco, thin cell layer explants, cell competence, shoot development, polarity     

ANATOMY OF THE SEED OF CONVOLVULUS ARVENSIS

Sripleng , Aksorn , (Kasetsart U., Bangkok, Thailand), and Frank H. Smith . Anatomy of the seed of Convolvulus arvensis. Amer. Jour. Bot. 47(5) : 386—392. Illus. 1960.–The anatropous ovule has a small, ephemeral nucellus covered by a massive integument. Shortly after fertilization, a lateral pouch develops from the upper portion of the embryo sac toward the dorsal side of the ovule and then downward. This leaves a partial integumentary septum in the base of the seed. The cellular endosperm is mostly absorbed by the embryo. Two—6 cell layers persist on all sides of the seed except below the cotyledons on the dorsal side where larger amounts persist. Over most of the seed the dermatogen develops into an epidermis that consists in part of groups of thick-walled elongate cells that produce the papillose appearance of the mature seed. The cells beneath the dermatogen divide periclinally and form 2 layers. The outer layer undergoes anticlinal divisions and differentiates a subepidermal layer of small, rectangular, thick-walled cells that become lightly lignified and suberized. The cells of the inner layer undergo some anticinal and periclinal divisions, elongate and differentiate as palisade sclerenchyma. The inner layers of the integument consist of parenchyma cells that are crushed and partially absorbed at maturity. The pad on the basal end of the seed, between the hilum and micropyle, is derived from a multiple epidermis that is differentiated into several layers of rectangular cells and a layer of palisade sclerenchyma. The subepidermal and palisade layers found over other parts of the seed dip beneath the pad.     

ADVENTITIOUS ROOT DEVELOPMENT FROM THE SEEDLING HYPOCOTYL OF LYCOPERSICON ESCULENTUM

Tomato seedlings five through ten days old were used for this investigation. Adventitious roots were initiated from the pericycle of the tomato hypocotyl. The position of adventitious root development was irregular in the rhizogenic hypocotyl; however, the cellular pattern of individual root development was very regular. Four layers of pericycle derivatives participated in root histogenesis and a bi- or triseriate endodermal cover was derived from the endodermis. Fluorescent microscopy showed that Casparian strips on the meristematic endodermal cell walls were not removed biochemically but were displaced around the root primordium by anticlinal divisions and cell enlargement. Casparian strips were not synthesized by endodermal cover cells. The emergent root had a typical three tiered or closed pattern of apical organization, and quiescent centers were present in all emergent roots longer than 0.5–0.6 cm.     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Lead accumulation and its translocation barriers in roots of Allium cepa L.—autoradiographic and ultrastructural studies

Abstract Lead migrating through the tissues of Allium cepa L. was found, by electron microscopy, autoradiography and other methods, to encounter at least three barriers to penetration. The layers of protoderm and hypodermic meristematic cells in the root meristematic zone and the layer of endodermis in the mature root zone were barriers to apoplastic transport. The central zone was a barrier to apoplastic and symplastic transport. It comprises the quiescent centre in the root meristem and the central part of the root cap. The cells of the deepest ground meristematic tissue layers seemed to act as a barrier, which keeps lead away from the procambium. Lead accumulated in roots but it was not uniformly distributed between their various tissues. The largest amount of lead accumulated both in ground meristematic and cortex tissues.     

Identification of specific plant nucleolar phosphoproteins in a functional proteomic analysis

The soluble fraction of nuclear proteins is a functionally significant fraction, since it has been shown that it contains ribonucleoproteins active in nuclear RNA metabolism. The aim of this work was to detect variations associated with cell proliferation, by comparing two-dimensional proteomes obtained from the soluble fractions of onion nuclei isolated from actively proliferating root meristematic cells versus nonmeristematic root cells. In particular, we have studied the physicochemical features of the major nucleolar protein NopA100, a highly phosphorylated, nucleolin-like protein. A total of 384 spots were quantified in meristematic nuclei, while only 209 were detected in nonmeristematic nuclei. The comparison of both proteomes resulted in the determination of specific spots for each proliferative state and those which were common to both cases. Furthermore, among these latter, we could discriminate quantitative differences. Interestingly, well-known nucleolar proteins, such as RNA polymerase I, B23 and the nucleolin-like protein NopA100, were significantly increased in proliferating cells. Western blots with anti-NopA100 antibody demonstrated 26 spots in the meristematic sample. All the spots detected were clustered at 100 kDa and were distributed through an isoelectric point (pI) range of 4.3-6.6. In contrast, only seven spots were found in the extract from nonmeristematic nuclei, and the pI range was shortened to 4.8-6.1. These results indicate that the state of NopA100 phosphorylation correlates with the degree of nucleolar activity, i.e. the protein is more highly phosphorylated in cycling cells. We have also analyzed the bidimensional silver staining of the nucleolar organizing region (Ag-NOR) pattern of the soluble nuclear fraction in order to identify plant cell phosphoproteins that are considered to be markers of proliferation. These experiments demonstrated that NopA100, the onion, nucleolin-like protein, is an Ag-NOR protein. In addition we found that the plant homologue of the vertebrate nucleolar phosphoprotein B23 migrated as two clusters of acidic spots, 43 and 42 kDa respectively in molecular mass. The differences between these features and those described for mammalian cells is discussed. Our results demonstrate that the use of protein fractionation procedures with functional significance and the location of candidate spots by indirect techniques are advantageous, complementary methods to random selection procedures for proteomic studies involving further mass spectrometry analysis.     

Histological study of initiation and development in vitro of adventitious roots in minicuttings of apple rootstocks of M 26 and EMLA 9

Apple rootstocks M 26 and EMLA 9 'COST' shoots propagated in vitro were used for the histological study of initiation and development of adventitious roots after a brief induction pretreatment. The results show that there are differences in mode and timing of initiation and development of adventitious roots between the two varieties. In M 26, adventitious roots were directly initiated from the derivatives of the cambium, some of which were immediately transformed into meristemoids in situ 36 h after pretreatment. Subsequently, meristemoids differentiated into root primordia. Development of adventitious roots were completed when they emerged at the surface of stem bases 10 days after pretreatment. In EMLA 9, before the meristemoids formed, internal cell files were formed by continuous divisions of cambial cells. The cells were regularly arranged in files external to the cambium. On the fourth day after pretreatment, some cells in the outermost layers of these files became meristematic, started to divide and turned into meristemoids, which differentiated into root primordia. The cells of the files between the root primordium and the cambium were transformed into vascular tissues which connected the vascular systems of the adventitious roots and stems.