Anticytokinin effects on in vitro response of embryogenic and nonembryogenic genotypes of Dactylis glomerata L.

The effect of triazine and carbamate type of anticytokinins on in vitro response of embryogenic and nonembryogenic genotypes of orchardgrass (Dactylis glomerata L.) was studied. Both compounds stimulated callus production. Anticytokinins influenced both the efficiency of somatic embryogenesis and frequency of embryoid formation.     

An acidic esterase as a biochemical marker for somatic embryogenesis in orchardgrass (Dactylis glomerata L.) suspension cultures

 The isoenzyme pattern of esterases (EC 3.1.1.2) secreted into the medium of orchardgrass (Dactylis glomerata L.) embryogenic suspension cultures during defined stages of somatic embryogenesis was compared with that of non-embryogenic suspension cultures during unorganised cell proliferation. Isoelectric focusing revealed the presence of 7–14 predominantly acidic isoforms. Comparison with the corresponding cell-wall isoenzyme pattern showed minor, mainly quantitative differences. The pattern of intracellular soluble esterases did not change markedly during somatic embryo development. A unique esterase whose migration in two-dimensional gel electrophoresis corresponds to an apparent molecular mass of 36 kDa and pI=3.8 was detected only in embryogenic cultures at very early stages of development. Since this isoform appeared long before morphological changes had taken place, it could possibly be used as a biochemical marker for embryogenic potential in D. glomerata L. suspension cultures. Received: 6 June 2000 / Revision received: 17 July 2000 / Accepted: 17 July 2000     

Development and characterization of embryogenic suspension cultures of pecan

A method for the establishment and proliferation of developmentally stable, embryogenic suspension cultures in pecan is described, and the growth and development of cultures characterized. Suspension cultures were generated from somatic embryos derived from zygotic embryo cotyledon explants induced on a solidified medium with naphthaleneacetic acid. Cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. The suspensions consisted of a mixture of globular stage embryo-aggregates, freely suspended globular embryos and pre-globular stage embryo masses. Culture growth and proembryo production were evaluated with respect to several liquid media and pH conditions. Significant differences in growth and productivity were observed between cultures. Pre-globular stage embryo masses collected on filter paper and overlaid on solidified medium continued ontological development and converted into plants. Thus a method has been developed for pecan suspension culture, which presents a major improvement in embryogenic tissue culture within the Juglandaceae. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

Summary Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division. divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all.     

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5–10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.Abbreviations B5 medium according to Gamborg et al. (1968) - SH-x medium according to Schenk and Hildebrandt (1972) supplemented with x M dicamba - dicamba 3,6-dichloro-o-anisic acid - KM-8p medium 8p of Kao and Michayluk (1975)     

Plant regeneration from embryogenic suspension cultures ofCamellia Japonica

Summary Two methods (I and II) for somatic embryo production from embryogenic suspension cultures ofCamellia japonica are presented. Method I, embryogenic suspension cultures, was established from suspension cultures initiated from leaf-derived callus. These cultures were maintained by reducing agitation and increasing subculture interval. Induction of somatic embryogenesis was achieved in MS28 medium, 6, 12, 24, and 36 mo. after culture establishment. Embryo production decreased after 1 yr of culture. Method II, suspensions of single embryogenic cells and proembryos, was obtained from leaves cultured in liquid MS13 medium 6 wk after culture initiation. Embryo production was 23 embryos/ml. Germination of cell suspension-derived embryos on MS56 medium was 16.7 % (±4.2%) for method I, and 35.4% (±5.1%) for method II. The embryos germinated into plantlets with 0 to 7 axillary shoots.     

Embryogenesis specific protein changes in birch suspension cultures

Two cell lines of birch (Betula pendula Roth.), one potentially embryogenic given the right inductive conditions and one which never has shown any embryogenic capacity, were both subjected to conditions inductive and non-inductive for somatic embryogenesis. Cells from these treatments were harvested at intervals over a 3-week period and washed with salt solution to wash off proteins loosely attached to the cell walls. The remaining cells were either freeze-dried whole or the cell walls were isolated. The extracted proteins from these three cell preparations were separated by one-dimentional SDS-polyacrylamide gel electrophoresis and detected by silver staining. Proteins specific for embryogenic cultures under inductive conditions were found in samples from the whole washed cells, whereas in the samples from isolated cell walls and “cell washings”, certain proteins seemed to disappear when the cells entered the embryogenic state. The changes in protein patterns were evident 24 h after the medium has been changed to embryo-production medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Composition of embryogenic suspension cultures of Ipomoea batatas Poir. and production of individualized embryos

Embryogenic suspension cultures of Ipomoea batatas Poir. contain heterogeneous populations of discrete cellular units. In order to optimize embryo production, a study was conducted to identify the embryogenic fraction of such cultures. Suspension cultures were fractionated with sieves of 1000, 710, 500, 355, 250, 180, 125, 90 and 63m mesh openings and the composition of each fraction was determined. Cellular units larger than 355 m were primarily calli and made up 75% of the total mass of cultures in the stationary phase of growth. These calli were composed of embryogenic and non-embryogenic subunits, and 98% of the embryogenic subunits measured 355–1000 m. Calli and embryogenic calli subunits produced clusters of embryos at various stages of development upon transfer to liquid or solidified media without 2,4-D. The 125–355 m fraction of suspension cultures was composed of cell aggregates of which 20% were embryogenic. The embryogenic cell aggregates produced single globular embryos upon transfer to liquid media containing 0 or 1 M 2,4-D. The 63–125 m fraction of suspension cultures contained only 2% of embryogenic cell aggregates. It can be inferred from our results that the embryogenic fraction of cultures was essentially represented in calli, and that proliferation of the embryogenic fraction occurred through the separation of embryogenic cell aggregates from larger calli when cultures approached their stationary growth phase.Abbreviations and definitions cellular units single cells, cell aggregates, and calli - cell aggregates discrete associations of cells - calli association of cell aggregates - embryogenic cell aggregates yellow aggregates of cytoplasmic cells which have the potential to produce embryogenic calli or embryos [3] - non-embryogenic cell aggregates white aggregates of vacuolated cells [3] - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid     

Evaluation of a plant regeneration test of embryogenic cell suspension cultures of rice (Oryza sativa L. cv Taipei 309)

Summary In this paper we discuss the changes in the number of plants developed per embryogenic mass, the percentage of embryogenic masses that developed plants, the percentage of plant regeneration, and the index of vigor of regenerated plants for embryogenic masses that were induced from daily samples of a cell suspension culture of rice (Oryza sativa L. cv. Taipei 309). The results indicate that embryogenic masses that matured first and were induced from freshly subecultured cell suspension culture had the highest values for the four parameters. An index of vigor of the regenerated plants was defined.     

Monoclonal antibody against a cell wall marker protein for embryogenic potential of <Emphasis Type="Italic">Dactylis glomerata</Emphasis> L. suspension cultures

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.     

Effect of culture period and cell density on regrowth following cryopreservation of embryogenic suspension cultures of Norway Spruce and Sitka spruce

Changes in cryotolerance, sedimented cell volume and dry weight were determined during a 21-day culture period for embryogenic suspension cultures of Norway spruce (Picea abies) and Sitka spruce (Picea sitchensis). Maximum cryotolerance was obtained for both species when the cultures were harvested in the phase of stationary growth in terms of dry weight. For P. abies, the culture period that resulted in maximum cryotolerance was similar to the culture period that resulted in maximum formation of mature embryos after 10 weeks of maturation. The initial cell density of the P. abies cultures is an important factor in affecting regrowth after cryopreservation and it was found that adjustment of the sedimented cell volume to 50% (v/v) resulted in maximum regrowth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differences in compounds released by embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

Media from embryogenic and non-embryogenic cell suspension cultures were analysed for protein content, electrophoretic protein patterns, glycoproteins and activity of peroxidases and β-glucosidases in order to characterize the physiological status of the cultures. On a dry mass basis the amount of extracellular proteins per cell was greater in embryogenic suspensions than in non-embryogenic suspensions. Non-embryogenic suspensions contained unidentified slimy compounds which were not present inembryogenic cultures. The extracellular Concanavalin A-specific glycoproteins gave different isoelectric focussing patterns and thus enabled embryogenic and non-embryogenic cultures to be differentiated. The extracellular peroxidase activity per cell dry mass was far greater in embryogenic than in non-embryogenic cultures. The isoenzymes differed in number and composition of the anionic bands. β-glucosidases were found in the same range of activity in both culture types, but the time course of enzyme activity during cultivation was significantly different. In the embryogenic culture the activity was correlated with dry mass increase, whereas in the non-embryogenic suspension the activity reached maximum during the linear growth phase. Polyphenoloxidase which was recently recognized as an intracellular marker for embryogenic stages was not released into culture media. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nutrient uptake and growth of an embryogenic and a non-embryogenic cell line of birch (Betula pendula Roth.) in suspension culture

The nutrient uptake of an embryogenic and of a non-embryogenic cell line of birch (Betula pendula Roth.) during cell growth and embryo production was studied in suspension culture. The embryogenic and non-embryogenic cell suspensions grew differently in the same medium. The non-embryogenic cell line started to grow without any lag period after the inoculation. It rapidly hydrolyzed sucrose in the medium to glucose and fructose and consumed the glucose as carbon source. The concentration of fructose in the medium decreased only after the depletion of glucose. The embryogenic cell line also rapidly hydrolyzed the sucrose to glucose and fructose, but the monosaccharides were consumed only after the embryos started to germinate after three weeks of culture. Both monosaccharides were then taken up at the same rate.     

BA enhances the germination of oil palm somatic embryos derived from embryogenic suspension cultures

Embryogenic suspension cultures of oil palm ( Elaeis guineensis Jacq.) allow mass propagation of somatic embryos; however regeneration rates are low. Histological observations have revealed that shoot development might be limited by the absence of a caulinary meristem. The addition of 6-benzyladenine during development was found to induce shoot apex differentiation and thus increased germination rates, by up to 70%. However, multiple shoot formation was a consequence of a longer period of cytokinin supply during the development of the embryo. In contrast, a short period of culture on medium with 6-benzyladenine at the begining of embryo development was found to result in single shoot production.     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

Cryopreservation of embryogenic suspension cultures of Cyclamen persicum Mill.

We have developed a simple protocol for the cryopreservation of embryogenic suspension cultures of Cyclamen persicum. Embryogenic suspension cultures in the linear growth phase 7–10 days after subculture were used for cryopreservation. Of the different cryoprotectants tested during a 2-day pre-culture, 0.6 M sucrose resulted in the highest re-growth rates of 75%. An additional pre-treatment with 0.6 M sucrose and 10% DMSO (dimethylsulfoxide) for 1 h also positively affected re-growth. Microscopic studies on viability revealed that only few small embryogenic cells survived cryopreservation, while vacuolated single cells died. Experiments in which the duration of the pre-culture period—i.e. the length of time the embryogenic suspension cells were exposed to 0.6 M sucrose—was varied showed that 2–4 days was the most optimal exposure time to 0.6 M sucrose. Callus re-grown after cryopreservation showed growth rates similar to that of unfrozen callus and regenerated even higher numbers of somatic embryos than unfrozen callus.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DM Dry mass - DMSO Dimethylsulfoxide - FDA Fluorescein diacetate - FM Fresh mass - 2IP 6-(,-Dimethylallylamino)purine - LN Liquid nitrogen - rpm Rounds per minute - SCV Sedimented cell volume     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

AC electrokinetic characterisation and separation of cells with high and low embryogenic potential in suspension cultures of carrot (Daucus carota)

Suspension cultures of Daucus carota L. were established, and cells with embryogenic potential were separated from those without by density gradient centrifugation in Ficoll at different stages in the growth curve. In order to obtain information about the electrical properties of individual cells, electrorotation spectra of single plant cells from different fractions were measured before and after induction of embryogenesis. The data were analysed using models based on Maxwell–Wagner's theories of interfacial polarisation. It was found that the denser cells had a higher embryogenic potential, a darker appearance and a higher internal conductivity (>1 S m^–1) than the less dense cells, which had less or no embryogenic potential and a lower internal conductivity (<1 S m^–1). Modelling the dielectrophoretic (DEP) response on the basis of the electrorotation data suggested that separation of cells with high embryogenic potential may be achievable in the frequency range 1–10 MHz. Actual dielectrophoretic separation of cells with high embryogenic potential from suspensions in which embryogenesis had not yet been induced was achieved using steric as well as hyperlayer dielectrophoretic Field-Flow Fractionation (DEP-FFF).     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.