Non-embryogenic Cells Research Articles

Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata 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.

Cellular behavior in embryogenic and non-embryogenic sugar beet calluses

The present study discusses the results of cytological studies of two kinds of sugar beet callus, i.e., embryogenic and non-embryogenic tissues. The calluses were produced through culture of secondary leaves on Murashige and Skoog medium containing two hormone combinations. One week after transfer of calluses onto fresh medium, their cells were viewed using electron microscopy and an image analyzer. Observations showed that cells of the two callus types had considerable differences in cell structure and various organelles. Of note were the high amount of polyploidization, rough endoplasmic reticulum, polysome, poly-nucleolus, and incomplete cell wall together with abnormal partitioning in non-embryogenic cells, as compared to embryogenic cells. In contrast, vacuolation of cytoplasm, perfect cell wall and partitioning structure, and the high proportion of nucleus/cytoplasm area were recognized in embryogenic cells.

Responses of meristematic callus cells of two Cynodon dactylon genotypes to aluminium

Responses to Al 3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al 3+ toxicity and resistance. A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al 3+ ion in culture media, which was supplied as 0.08–2.3 mM Al 3+ for 2–8 weeks. Increasing Al 3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells. Exposure to 0.8 mM Al 3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected. In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis. The Al-R cells appeared to exclude Al 3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R). The results showed that by studying the cellular responses to Al 3+ it is possible to discriminate between the Al-S and Al-R C. dactylon genotypes.

The Influence of Growth Regulators on Membrane Permeability in Cultures of Winter Wheat Cells

The effect of plant growth substances (IAA, 2,4-D, zeatin, kinetin, zearalenone) were studied on membrane properties of the cells of embryogenic (E) and non-embryogenic (NE) calli derived from immature inflorescences (inf) or embryos (emb) of winter wheat. Calli initiated from inflorescences show higher permeability. The ion leakage from cells of E calli was higher than from cells of NE calli. Growth regulators were used in concentrations of 2-30 mg/l (about 10-140 microM). All tested growth substances increased ion leakage from NE emb cells, IAA, zeatin and kinetin being most effective. In NE inf cells the effect of growth substances was similar as in NE emb, but much weaker. In E cells of both types (inf and emb) growth substances decreased ion leakage. Changes in the leakage of potassium and calcium ions were similar to those in total ion leakage. The uptake of labelled auxins (IAA and 2,4-D) was higher in NE cells (especially in NE inf) than in E cells. The endogenous level of IAA was higher in E cells than in NE cells and in inf cells than in emb cells. The importance of auxin in determining permeability of cell membranes is discussed.

SERK Gene Homolog Expression, Polyamines and Amino Acids Associated with Somatic Embryogenic Competence of Ocotea catharinensis Mez. (Lauraceae)

In the present work, we investigate the association of SERK gene homolog expression, polyamines (PAs) and amino acids related to putrescine synthesis (arginine and ornithine) and polyamines degradation (γ-aminobutiric acid) or S-adenosylmethionine synthesis (methionine), with the embryogenic competence in cell aggregates of Ocotea catharinensis Mez. (Lauraceae). Cell aggregates were cultivated during 7 days in woody plant medium (WPM) supplemented with 20 g l−1 sucrose, 22 g l−1 sorbitol, 400 mg l−1 glutamine and 2 g l−1 phytagel, and in Murashige and Skoog medium (MS) supplemented 20 g l−1 sucrose, 3 g l−1 activated charcoal, 2 g l−1Phytagel with and without 40 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). The cell aggregates cultivated in MS plus 2,4-D and in the WPM medium showed hybridization with a SERK gene homolog both in northern and in situ hybridization experiments. Cell aggregates cultivated in an MS basal medium, without 2,4-D, did not exhibit any hybridization signal to the SERK probe used, thus they were considered potentially non-embryogenic cells. In all three media only free polyamines were detected. The higher putrescine levels occurring in WPM callus were associated with a higher arginine and ornithine content, lower γ-aminobutiric acid level, and SERK homolog expression. Putrescine was also the major polyamine in the MS medium. In the MS plus 2,4-D medium, the levels of putrescine, spermidine and spermine were similar. Spermine exhibited similar and the lowest levels in all media. Spermidine intermediary levels occurred in the WPM and MS media. In cell aggregates methionine level was lowest in the MS plus 2,4-D medium, but similar in the MS and WPM media.

Phytosulfokine-α requires auxin to stimulate carrot non-embryogenic cell proliferation

Phytosulfokine-α (PSK-α), a peptidyl plant growth factor that was originally isolated from conditioned medium derived from asparagus mesophyll cell culture, stimulates cell proliferation in some plants in suspension culture. However, the physiological relationship between phytohormones and this growth factor in plants is not well understood. Using carrot ( Daucus carota L.) non-embryogenic cell culture, we investigated the stimulatory effects of PSK-α on the cell proliferation induced by different combinations and concentrations of auxin [2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), or naphthaleneacetic acid (NAA)] and PSK-α. Cell proliferation was not stimulated by PSK-α at high auxin concentrations, but was promoted at low auxin concentrations. There was no proliferation in the absence of auxin. These results indicate that PSK-α stimulates cell proliferation when cell division activity is low, and that auxin is required for this process. We also evaluated PSK-α production using medium derived from non-embryogenic cell culture with high or low concentrations of auxin (2,4-D, IAA or NAA) in a competition ELISA system. More PSK-α was produced at high auxin concentrations than at low auxin concentrations. These results suggest that auxin participates in PSK-α production.

Phytosulfokine-.ALPHA., a Peptidyl Plant Growth Factor, Stimulates Cell Cycle Progression in Carrot Non-Embryogenic Cells.

Phytosulfokine-.ALPHA., a Peptidyl Plant Growth Factor, Stimulates Cell Cycle Progression in Carrot Non-Embryogenic Cells.

The Role of auxin, pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa.

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.

The influence of phytohormones on zeta potential and electrokinetic charges of winter wheat cells.

The zeta potential measurements of protoplasts obtained from winter wheat cell culture and phospholipid liposomes were performed to determine the electrokinetic charge in a medium containing various phytohormones (kinetin, 2,4-D and zearalenone) in absence and in presence of 2 x 10(-5) MCa2+. Calli were induced from immature inflorescences (inf) and embryos (emb) and cultured to obtain non-embryogenic (NE) and embryogenic (E) cell tissues. All investigated phytohormones indicate ability to adsorb to the negatively charged surfaces (latex, L88 - model negative adsorption site) both in water solutions and at the presence of mannitol and buffer (MES). In biological systems (protoplasts and liposomes - prepared from phospholipids of protoplasts) the electrokinetic charges were dependent on the phospholipid and protein composition of cells. The influence of protein groups on electrokinetic charge was calculated from charge values of protoplasts and liposomes, assuming additivity of surface charges. The comparison of calculated charges for protoplasts and liposomes indicate that 2,4-D is better adsorbed to the phospholipid and proteins of NE cells whereas kinetin is bound to the phospholipid and protein sites of E calli. This effect may be connected with embryogenesis process, where non-embryogenic culture of wheat requires 2,4-D in the medium, and embryogenic culture requires cytokinin rather. Zearalenone binding is especially dependent on the kind of explant.

Flow cytometric analysis of cellulose tracks development of embryogenic Brassica cells in microspore cultures

Summary • Brassica napus microspore cultures are excellent systems for studying embryogenesis in plants because they are single celled, induced by a simple heat treatment and produce structures that are very similar to zygotic embryos. A method for detecting embryogenic cells that is based on a surface marker would be useful for studying the early events in this developmental process. • Since the production of a cellulosic cell wall is thought to be a characteristic of embryogenic cells in these cultures, a flow cytometric method for monitoring cells producing cellulose, after staining with calcofluor white, was developed. • Results showed that the induced cultures developed a unique population of cellulose-containing cells. On the basis of their morphology, these cells were identified as the embryogenic cells after flow cytometric sorting. • The study also documented the development of embryogenic and nonembryogenic cells in microspore cultures over a 4-d period on the basis of their cellulose content and size.

4-Hydroxybenzyl alcohol accumulates in suspension-cell cultures and inhibits somatic embryogenesis in carrot.

Somatic embryogenesis in carrot (Daucus carota L.) is strongly inhibited by certain factors that accumulate in culture medium of high-density cultures of embryogenic cells. We previously identified 4-hydroxybenzyl alcohol (4HBA) as one of the inhibitory factors. In this study, we analyzed the accumulation pattern of 4HBA in the cultures of carrot suspension cells. When somatic embryogenesis was induced by culturing embryogenic cells in phytohormone-free Murashige and Skoog medium at various initial cell densities, 4HBA accumulated in the culture medium. The concentration of 4HBA in high cell density cultures was higher than in low cell density cultures. The accumulation of 4HBA in high cell density cultures was rapid during the early days of culture. This rapid accumulation of 4HBA in high cell density cultures might result in the strong inhibition of somatic embryogenesis. The production of 4HBA decreased as the somatic embryos developed. In addition, embryogenic cells released larger amount of 4HBA into the culture medium compared with non-embryogenic cells. These results suggest that the production of 4HBA is both related to embryogenic competence and developmentally regulated during somatic embryogenesis.

The effects of glutamine of the maintenance of embryogenic cultures of Cryptomeria japonica

Embryogenic tissues of sugi (Cryptomeria japonica) were induced on a modified Campbell and Durzan (CD) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 600 mg l−1 glutamine, and subcultured in the medium of the same composition for over 1 yr. This resulted in a mixed culture of embryogenic and non-embryogenic cells. When embryogenic cells were isolated and cultured independently, their capacity to form embryogenic aggregates was lost. Thus, the non-embryogenic cells present within a mixed culture system were essential to the formation of embryogenic aggregates. When embryogenic tissues were isolated and cultured independently on a high glutamine-containing (2400 mg l−1) medium, dry weights and endogenous levels of glutamine increased, and the tissue could generate a large number of embryogenic aggregates. Amino acid analysis of embryogenic and non-embryogenic cells from the maintenance culture indicated a higher level of glutamine was present in the latter. The high endogenous level of glutamine in the non-embryogenic portion of mixed cell masses may be the supplier of glutamine for maintaining the embryogenic property of the tissues.

Proteins related with embryogenic potential in callus and cell suspensions of sugarcane (Saccharum sp.)

Previous results have shown that some proteins secreted in the culture medium are involved with the formation of embryogenic cells and can modify somatic embryo differentiation. Undifferentiated cell suspensions grown in the presence of 13 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and obtained from embryogenic and non-embryogenic callus were used to study these events in sugarcane plants (cv.PR-62258). The cell suspension growth curves were determined and soluble proteins were extracted from embryogenic and non-embryogenic callus and culture medium from cell suspensions. In embryogenic callus we detected 1.43 times more protein than in non-embryogenic callus and the electrophoretic protein patterns show specific polypeptides for both callus types. In embryogenic callus we detected a cluster of four polypeptides in the range of 38–44 kDa and another polypeptide of 23 kDa that were not observed in non-embryogenic callus. In nonembryogenic callus there is a 35-kDa polypeptide that was not detected in embryogenic callus. In the case of extracellular proteins, the medium from embryogenic cell suspensions contained four polypeptides of 41, 38, 34 and 28 kDa that were slightly detected in the medium from non-embryogenic cell cultures; we also detected a band at 15 kDa that could not be observed in the medium from non-embryogenic cell suspensions. These results suggest that the development of embryogenic callus and cell suspensions is related to the type and amount of intracellular proteins in the callus cells and to the secreted proteins from these cells into the medium.

Partial characterization of genes whose transcripts accumulate preferentially in cell clusters at the earliest stage of carrot somatic embryogenesis.

We attempted to identify genes that are preferentially expressed immediately after somatic cells divide to form cell clusters at the earliest stage of carrot somatic embryogenesis when they are not or barely expressed in non-embryogenic suspension-cultured cells in the presence of 2,4-D. Using the differential display technique, we isolated three cDNA clones, designated No. 43, No. 87 and No. 93. The No. 43 transcript was preferentially expressed in the earliest cell clusters, its level decreased drastically at the globular and heart-shaped and torpedo-shaped stages, and it was not detected in non-embryogenic suspension-cultured cells. No. 43 cDNA encoded a protein with homology to thaumatin-like proteins and the deduced positions of seven cysteine residues in the 63 amino acid sequence from the carboxyl terminus were identical to those in thaumatin-like proteins. The full-length nucleotide sequence of No. 93 cDNA was determined and its product was about 80% homologous to precursor of the 14 kDa proline-rich DC 2.15 protein of carrot at the amino acid level. However, the deduced amino acid sequence lacked the characteristic core of repeating Pro-X motifs found in DC 2.15. The No. 93 transcript accumulated preferentially in the earliest cell clusters but it was also detected at a low level in non-embryogenic suspension-cultured cells, unlike DC 2.15 transcripts that begin to accumulate in heart-shaped embryos before their level falls in torpedo-shaped embryos. No. 87 transcripts were expressed preferentially in the earliest cell clusters that has been incubated with 2,4-D but were also detected at a low level in suspension-cultured cells subcultured in the continued presence of 2,4-D. The No. 87 cDNA exhibited no significant homology to any sequences in databases.

Initial identification of a phosphoprotein that appears to be involved in the induction of somatic embryogenesis in carrot.

We examined changes in the absence versus presence patterns of phosphoproteins with respect to the acquisition of embryogenic competence during somatic embryogenensis in carrot (Daucus carota L.). To characterize a possible correlation between the induction of embryogenic competence and protein phosphorylation, we examined the patterns of protein phosphorylation in embryogenic cells (EC) and non-embryogenic cells (NC) that had lost the ability to form somatic embryos. Two-dimensional polyacrylamide gel electrophoresis and subsequent autoradiography revealed the presence of 31 phosphoproteins in EC but not in NC. Furthermore, when we examined the induction of somatic embryogenesis by certain stress compounds in the absence of phytohormones, we identified one specific phosphoprotein (ECPP-44). ECPP-44 was found to be induced in all treatments that resulted in embryogenic competence. The partial amino acid and nucleotide sequence of ECPP-44 shows partial homology to two dehydrins (ERD10 and ERD14) from Arabidopsis.

Acquisition of Desiccation Tolerance by Cultured Carrot Cells upon Ectopic Expression of C-ABI3, a Carrot Homolog of ABI3

To obtain direct evidence for the involvement of C-ABI3, a carrot ( Daucus carota L.) homolog of VP I/ABI3, seed-specific transcription factor, in the acquisition of desiccation tolerance, we prepared transgenic carrot non-embryogenic cells (NC) in which the C-ABI3 gene was expressed ectopically. Non-transgenic NC, in which expression of C-ABI3 was not detected, did not exhibit desiccation tolerance even after treatment with abscisic acid (ABA). In transgenic NC that expressed C-ABI3 , embryo-specific ABA-inducible genes ( ECP genes) were expressed upon ABA-treatment. Furthermore, the transgenic NC became desiccation-tolerant upon ABA-treatment, bur did not tolerate desiccation without ABA-treatment.

Cloning and Characterization of ECPP44, a cDNA Encoding a 44- kilodalton Phosphoprotein Relating to Somatic Embryogenesis in Carrot.

Somatic embryogenesis in Daucus carota can be induced by the treatment of shoot apices with various kinds of stress chemicals (Tachikawa et al., 1998). Using this system, we previously revealed the presence of a phosphoprotein (ECPP44) of which the phosphorylation was specific to embryogenic cells (EC), stress-treated shoot apices and somatic embryos, but not in non-embryogenic cells (NC). We then obtained a 141 by fragment of ECPP44 cDNA by RT-PCR with some primers based on the partial amino acid sequence. In this research, the fragment was used as a probe to screen the cDNA library which was compiled from somatic embryos. A full-length cDNA (925bp) corresponding to ECPP44 was isolated and sequenced. The putative amino acid sequence revealed that ECPP44 contained poly-serine consensus and nuclear targeting signal of Group II LEA genes. Phylogenetic analysis of the amino acid sequence encoded by ECPP44 showed that ECPP44 is a distant homology of the dehydrin family in carrot. ECPP44 is a single copy gene as evidenced by Southern blot analysis under high stringency. Northern blot analysis revealed that ECPP44 mRNA accumulates in EC, stress-treated and non-treated shoot apices, and somatic embryos, but not in NC. We discuss here the accumulation and/or phosphorylation of ECPP44 as it relates to the acquisition of embryogenic competence.

Inhibitory Effects of p-Hydroxybenzyl Alcohol on Somatic Embryogenesis in Carrot Cell Cultures.

Somatic embryogenesis in carrot (Daucus carota L.) is strongly inhibited by certain factors that accumulate in the medium of cultures with high embryogenic carrot cell density. We previously identified p-hydroxybenzyl alcohol as one of these inhibitory factors. In this study, we performed a detailed analysis of the effects of p-hydroxybenzyl alcohol on somatic embryogenesis. p-Hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos by suppressing cell division. The inhibitory effect on the development of globular embryos was stronger than that on the development of heart-shaped and torpedo-shaped embryos. In contrast, p-hydroxybenzyl alcohol had no effect on the proliferation of both undifferentiated embryogenic cells or non-embryogenic cells. These results indicate that p-hydroxybenzyl alcohol inhibits the division of cells in a way that is specific to the early stage of somatic embryogenesis and that plays an important role in the formation of somatic embryos.

Starch, Storage Protein and Triglyceride Accumulation and Respiration in Developing Embryos in Hevea brasiliensis

Summary Starch, storage protein and triglyceride contents and respiratory rate were studied in an embryogenic strain of Hevea brasiliensis during development of somatic embryos. For comparison, a non-embryogenic strain was also investigated. The development of somatic embryos was studied on a medium containing polyethylene glycol and abscisic acid, and a medium devoid of both. Somatic embryos were almost lacking on medium devoid of polyethylene glycol and abscisic acid: only 3% of torpedo embryos versus 40% on medium containing these substances. Storage product accumulation in somatic embryos was observed on the medium containing polyethylene glycol and abscisic acid but not on the medium devoid of both. It took place in torpedo embryos; this could explain why it was absent on medium devoid of polyethylene glycol and abscisic acid. In torpedo embryos, storage proteins and triglycerides were accumulated at the same time, followed by starch deposition. Starch, storage proteins and triglycerides represented respectively 4.4 %, 36.1% and 59.5% of the total reserves accumulated. In non-embryogenic cells, starch, storage protein and triglyceride levels were similar whatever the culture conditions. The respiration rate was greater in embryogenic and non-embryogenic cells cultured on the medium containing polyethylene glycol and abscisic acid than on the medium devoid of both. This stimulation of respiration could be linked with greater osmotic pressure of the medium containing polyethylene glycol. Also, when torpedo embryos were formed, respiratory rate decreased and accumulation of storage products was observed.

Somatic embryogenesis from immature and mature zygotic embryos ofCryptomeria japonica I: Embryogenic cell induction and its morphological characteristics

Embryogenic cells (ECs) of sugi (Cryptomeria japonica D. Don) were induced from immature and mature zygotic embryos cultured on different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D)-containing modified Campbell and Durzan medium. The rate of induction of ECs varied depending on the stage of embryos collected. The highest percentage of induction (35%) was obtained with immature zygotic embryos collected July 18 and July 30, 1997, when 1 μM of 2,4-D was added to the induction medium. The ECs easily proliferated when subcultured in a medium of the same composition as the induction medium within 3 weeks. Morphological characteristics of nonembryogenic cells and embryogenic cells of different developmental stages were studied under an inverted fluorescence microscope.