Inhibition of ethylene production and stimulation of carrot somatic embryogenesis by salicylic acid

The effects of salicylic acid (SA) and other phenolic compounds, acetylsalicylic acid (ASA), benzoic add (BA) and sulfosalicylic acid (SSA), on ethylene production and somatic embryogenesis by carrot (Daucus carota L.) cell cultures were studied. SA and ASA, at concentrations of 10 μM and 100 μM, significantly stimulated somatic embryogenesis and effectively inhibited ethylene production by carrot cell suspension cultures. The observed increase of embryo number was proportional to the inhibition rate of ethylene production. However, BA and SSA affected neither ethylene production nor somatic embryogenesis. The role of SA in somatic embryogenesis is discussed.     

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.     

Unusual patterns of somatic embryogenesis in the domesticated carrot: developmental effects of exogenous auxins and auxin transport inhibitors

The effects of various exogenous auxins and polar auxin transport inhibitors on somatic embryogenesis in carrot cultures were investigated. Indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid do not disrupt the sequence or the polarity of individual stages in embryo development, but tend to cause developing embryos to revert to undifferentiated callus, with increasing frequency in later embryo stages. The transport inhibitors, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid, block morphological transitions to the subsequent stage; for example, they cause the formation of enlarged globular and oblong embryos. Heart embryos in these treatments usually develop additional lateral growth axes. These results shed light on the role of auxin and its polar transport in somatic embryogenesis.     

Co-culture with Daucus carota somatic embryos reveals high 2,4-D uptake and release rates of Arabidopsis thaliana cultured cells

By means of co-culture in growth regulator-free medium we analysed whether factors secreted into the medium of Daucus carota (carrot) somatic embryo cultures would be able to overcome the developmental arrest of globular Arabidopsis thaliana somatic embryos. Instead of Arabidopsis embryogenesis being promoted the development of carrot somatic embryos was inhibited at the globular stage in the presence of Arabidopsis suspension culture aggregates with attached globular embryos. Several experiments showed that this was due to the release of previously accumulated 2,4-D by the Arabidopsis cultures. (1) In addition to arresting carrot embryogenesis, co-culture with Arabidopsis cell suspensions also induced callus formation on Arabidopsis root segments. (2) Both effects only occurred with Arabidopsis suspensions grown in the presence of 2,4-D and not with those grown in the presence of NAA, demonstrating that Arabidopsis is not segregating a “general” inhibiting factor. (3) Both effects could be prevented by either binding 2,4-D to active charcoal or by washing it away by changing the medium daily. (4) Uptake of 2,4-D into Arabidopsis cells during culture in 2,4-D containing medium and subsequent release of 2,4-D after transfer to growth regulator-free medium was measured. (5) These low levels of released 2,4-D (0.2– 0.5 μm) could mimic the observed effects. Taken together these data suggest that the high intracellular 2,4-D content of Arabidopsis cultures may interfere with Arabidopsis somatic embryo development beyond the globular stage. Received: 13 November 1997 / Revision received: 2 February 1998 / Accepted: 16 November 1998     

Restoration of embryogenic competence in repeatedly subcultured carrot cell lines

Summary The production of somatic embryos from carrot suspension cultures invariably decreases through simple, repeated subculturing. Extracellular, concentrated compounds extracted from already established embryo culture not only recovered the embryogenic capability, but also accelerated the embryo production as much as two-fold (up to 1600 embryos/ml) compared with that of a control culture. Sugars, which were only a small portion of the total concentrate, were excluded as possible causative factors. It is likely that a protein fraction that is generated directly by competent, embryogenic cultures is important for the restoration of embryogenic potential.     

Effect of the tetrapeptide RGDS on somatic embryogenesis in Daucus carota

The present work examines the requirement for RGD-binding sites, such as those in the plasma membrane protein integrin during somatic embryogenesis in carrot ( Daucus carota L. cv. Scarlet nantes). It is possible to assay for this requirement by competitively inhibiting binding of the site to the natural RGD-containing ligand by adding an excess of synthetic RGDS. We found that treatment of carrot callus cultures with RGDS (2.7 m M ) inhibited the development of a normal shoot pole in carrot somatic embryos. The structures that formed contained separate zones of highly cytoplasmic and vacuolate cells and no evidence of embryonic organization, although occasionally a root-like structure was observed. If the aspartic acid residue in the peptide was replaced by a chemically similar amino acid (glutamic acid), the resultant somatic embryos were indistinguishable from those developing in untreated cultures. These effects are similar to those reported in animal systems where the protein receptor involved has been identified as integrin. Our results are thus consistent with the conclusion that a binding site for a motif similar to the integrin-binding site in a variety of animal extracellular matrix proteins exists in plants and appears to be important in somatic embryo development in carrot.     

Analysis of the rolC promoter region involved in somatic embryogenesis-related activation in carrot cell cultures.

In cell cultures of carrot (Daucus carota L.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Internal deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos. When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.     

A secreted peptide growth factor, phytosulfokine, acting as a stimulatory factor of carrot somatic embryo formation

Somatic embryogenesis of the carrot (Daucus carota L.) depends on a set of factors, some of which accumulate in culture medium (conditioned medium, CM). When embryogenic cell clusters were transferred to an embryo-inducing medium, addition of CM derived from somatic embryo culture markedly stimulated somatic embryo formation. The active principles were purified using a simple bioassay system and identified to be phytosulfokines (PSKs), sulfated oligopeptide growth factors originally isolated from a CM derived from asparagus (Asparagus officinalis L.) mesophyll culture. Quantification studies using a competition ELISA system employing an anti-PSK-alpha polyclonal antibody showed that PSK production might be related to growth of cells, rather than development of somatic embryos. Thus the stimulatory effect of PSK on somatic embryo formation might be due to promotion of cell proliferation.     

Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture

Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 × 10⁻⁴M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis. Received: 22 April 2000 / Accepted: 8 June 2000     

Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.     

Stimulation ofDaucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel

The effects of Co²⁺ and Ni²⁺ on ethylene production and somatic embryogenesis by carrot (Daucus carota L.) cell cultures were studied. At concentrations of 10 M to 50 M, CoCl₂ effectively inhibited ethylene production by embryogenic cultures and significantly stimulated somatic embryogenesis. The observed increase of embryo number was proportional to the inhibition level of ethylene production. However, CoCl₂ had no effect when Ethephon was supplied. Nickel also reduced ethylene production, but to a slightly lesser extent than CoCl₂, bringing about a lower increase in the number of somatic embryos. The role of ethylene on somatic embryogenesis is discussed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - SAM S-adenosyl-methionine     

Synchronized somatic embryo development in embryogenic suspensions of Fraxinus Angustifolia

Summary The synchronization of somatic embryo development in embryogenic suspension cultures is a crucial step in taking advantage of somatic embryogenesis for high production potential and reduction of unit cost through automation. In the present study, a synchronous somatic embryogenic system was developed for Fraxinus angustifolia suspension cultures. High cell density, 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid proved essential for the establishment and maintenance of suspension cultures. Low cell density, BA and 1-naphthaleneacetic acid enhanced somatic embryo development. Cell and cell cluster fractionation by density gradient centrifugation in Ficoll solution proved useful for separation of subpopulations with differing potentials for embryo development. A synchronous development of somatic embryos at high frequency was achieved only from the heaviest cell population.     

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes     

The effect of glutathione on development in wild carrot suspension cultures

The role of reduced and oxidized glutathione in plant development was investigated using wild carrot suspension cultures. Concentrations of GSH are lower in developing than in proliferating carrot cultures. Addition of 0.3 mM buthionine sulfoximine (a glutathione synthesis inhibitor) to developing cultures decreased the cellular GSH levels and enhanced somatic embryogenesis while addition of 0.6 mM GSH increased the cellular GSH levels and inhibited embryogenesis. Additions of GSH and buthionine sulfoximine to developing cultures also indicated that buthionine sulfoximine is acting specifically to lower GSH levels and not through some nonspecific toxic effect. These results provide evidence that the levels of GSH are important in determining whether carrot cells develop into somatic embryos or grow proliferatively.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Improvement of somatic embryogenesis in wild cherry (Prunus avium). Effect of maltose and ABA supplements

Three different types of morphogenesis were identified in embryogenic cultures of Prunus avium grown on a proliferation medium containing 0.54 μM NAA, 0.46 μM kinetin and 0.44 μM BA: a friable hyperhydric callus, repetitive embryogenesis and an embryogenic tissue. Translucent and white somatic embryos were produced from the three types of morphogenesis but mainly from the embryogenic tissue. These somatic embryos showed histological and cytological teratological features such as highly differentiated cells with shrunken cytoplasm and destructured nuclei. For the four lines studied, somatic embryo production was improved by transferring the embryogenic tissue to developmental media without auxin and cytokinin but supplemented with maltose alone or maltose and 10 μM ABA. Three weeks after transfer, the line showing the most embryogenesis produced 1404 somatic embryos per gram of embryogenic tissue. A concentration of 263 mM maltose significantly increased the number of white somatic embryos for L 10 line, while translucent somatic embryo production was improved by 88 mM maltose for L 16 line. The combination of maltose and ABA produced different effects with each line. When used with 88 mM maltose, 10 μM ABA significantly increased white somatic embryo production for two lines but decreased the production for one line. When combined with 263 mM maltose, ABA had no effect on white somatic embryo production but significantly decreased the number of translucent somatic embryos. Cells of white somatic embryos contained protein storage reserves and numerous lipid bodies, while those of translucent embryos did not contain storage reserves or lipid bodies. After a two-month cold treatment conversion rate of white and translucent somatic embryos reached 8.5% and 35.2% respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification from conditioned medium and chemical identification of a factor that inhibits somatic embryogenesis in carrot

Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells when the cell density is high. The inhibition is caused by factors that are released by cells into the medium of such cultures. In this study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of carrot cells. The inhibitory factor with the strongest apparent activity was purified by fractionation with ethylacetate, chromatography on an octadecylsilyl (ODS) silica gel-column and HPLC. The inhibitory factor had a single peak of absorbance at 280 nm and was identified as 4-hydroxybenzyl alcohol by mass spectrometry and 1H- and 13C-NMR spectroscopy. Authentic 4-hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos at a concentration equal to that in high-cell-density cultures. These results suggest that 4-hydroxybenzyl alcohol is a major factor that accumulates in high-cell-density cultures of carrot cells and inhibits somatic embryogenesis.     

Relationship between production of carrot somatic embryos and dissolved oxygen concentration in liquid culture

To evaluate the relationship between somatic embryogenesis and dissolved oxygen concentration, somatic embryo cultures of carrot (Daucus carota L.) were cultured under various dissolved oxygen concentration levels (bubble free aeration with 4%, 7%, 20%, 30%, and 40% oxygen in flasks). The system used allows dissolved oxygen concentration control without bubble aeration or mixing speed modification. The total number of somatic embryos was not affected by the dissolved oxygen (DO) concentration tested. Even if globular-stage embryos were induced at a low level of oxygen aeration, heart-stage embryo formation was still repressed. Oxygen enrichment (20%, 30% and 40% oxygen) enhanced torpedo and cotyledonary-stage embryo production. The oxygen-enriched aeration was effective in promoting the growth of the late developmental stages. Sugar consumption did not increase when the oxygen concentration was enriched above the ambient level. The number of heart-stage embryos increased as oxygen concentration increased up to the 7% level, while above the 20% level no change in production was observed. The production of cotyledonary-stage embryos was directly related to oxygen concentration. These results support that oxygen-enriched aeration provides oxygen to the low oxygen areas in somatic embryo. After the heat-stage embryos, which were grown at the 7% level were transferred to a flask with ambient, they developed an elongated root part and eventually grew to normal plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Increased ethylene and decreased phenolic compounds stimulate somatic embryo regeneration in leaf thin section cultures ofDoritaenopsis hybrid

The effects of endogenous levels of ethylene and phenolic compounds on somatic embryogenesis, medium-browning, and peroxidase activity were evaluated in thin section cultures ofDoritaenopsis. Cultures were maintained for 8 weeks with four different treatments: i) thick leaf segment culture, ii) thin leaf section culture, iii) thin leaf section culture with ventilation, or iv) thin leaf section culture after expiants were first washed. Expiants cultured in closed vessels produced a larger number of somatic embryos than those reared in the ventilated vessels. This enhanced formation confirmed the greater involvement of accumulated ethylene under non-ventilated conditions, because wound-induced tissues from thin leaf sections normally release high level of ethylene. When expiants were washed in the liquid medium and inoculated on the same solid medium, somatic embryo production was 1.7 and 18.5 times higher than in the thin section cultures and thick segment cultures, respectively. Reducing the level of phenolics in expiants at the initial stage of culturing apparently stimulated this embryo regeneration.