Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.     

Plant regeneration from mesophyll protoplasts of the tree legume Pithecellobium dulce benth

Conditions were developed for the isolation, culture and regeneration of mesophyll protoplasts of the tree legume, Pithecellobium dulce Benth. The presence of 2,4-dichlorophenoxyacetic acid (2,4-D) was essential to induce initial cell divisions and addition of naphthaleneacetic acid (NAA) improved the response. Sustained division and cell colony formation were achieved from the protoplasts cultured in a modified KM8P medium containing 2,4-D (2.3 μM), NAA (3 μM) and benzyladenine (BA) (2.3 μM). Dilution of the osmotica included in the protoplast culture medium was necessary to induce sustained proliferation of the protoplast-derived cells. Differentiation of shoots from the protoplast-derived calli occurred on Murashige and Skoog (MS) medium supplemented with BA (5 μM) and indole-3-acetic acid (1 μM). Omission of 2,4-D from the culture medium, after the initial 2 weeks of protoplast culture, was obligatory to induce shoot morphogenesis.     

Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts

A xyloglucan oligosaccharide was isolated from cell walls of Daucus carota L. suspension-cultured cells. From analytical data (gel-permeation chromatography, thin-layer chromatography, monosaccharide analysis, methylation analysis) it can be concluded that this oligosaccharide preparation consists mainly of a nonasaccharide known as XG9 (Glc₄Xyl₃GalFuc). This nonasaccharide showed excellent “anti-auxin” properties in the pea-stem bioassay, with 80% inhibition of 2,4-dichlorophenoxyacetic acid (2,4-D)-induced longitudinal growth of etiolated pea stem segments at concentrations of 1-0.1 nM. Applied in nanomolar concentrations to protoplasts regenerating in a medium containing 4.52 μM 2,4-D, the nonasaccharide influenced the viability of the protoplasts and the activities of glycan synthases in vitro. The effects were similar to those achieved by the omission of 2,4-D from the regeneration medium. The composition of the regenerated cell wall was not changed significantly by the use of 2,4-D-depleted medium or the addition of XG9 to 2,4-D-containing medium.     

Partial Synchronization of Carrot Cell Culture by Auxin Deprivation

A strain of carrot cells (Daucus carota cv. Kintoki) grew exponentially in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D, 1 mg/1) with a doubling time of about 2 days. When those cells were transferred to a medium lacking 2,4-D, they continued to grow at almost the same rate for about a week. When the cells were again transferred to the auxin-free medium, the rate of cell division gradually decreased. After the cell division had ceased, cells were returned to the ordinary 2,4-D medium. A burst of cell divisions occurred after about 2 days. Timing of DNA synthesis and of mitosis suggested that the cells had been arrested at G₁ phase. In a medium containing indoleacetic acid instead of 2,4-D, the auxin was rapidly degraded and the culture was similarly synchronized as in the auxin-omitted medium.     

Protein synthesis associated with quiescence and senescence in auxin-starved pear cells

Pear fruit cells (Pyrus communis L. cv Passe Crassane) stopped dividing when subcultured in a bioreactor under auxin starvation in the presence of 0.37 molar mannitol. The cessation of cell division was preceded by the accumulation of a specific basic polypeptide of 24 kilodalton. Readdition of 2.3 micromolar 2,4-dichlorophenoxyacetic acid (2,4-D) neither caused a resumption of cell division nor depressed the accumulation of this polypeptide. Under complete auxin starvation, cells began to die at day 18. In vivo radioactive labeling of proteins followed by two-dimensional electrophoresis showed that during auxin starvation the synthesis of some polypeptides including the 24 kilodalton one (referred to as homeostasis-related proteins, HRPs) was decreased while the synthesis of some others (referred as senescence-related proteins, SRPs) was increased. Readdition of 2.3 micromolar 2,4-D postponed the onset of cell death by 10 to 15 days while supplementation with 7.6 micromolar abscisic acid advanced cell death by 8 days. Two-dimensional analysis of protein synthesis indicated that both hormones interact on the synthesis of these two groups of polypeptides. The levels of most HRPs were maintained or increased in the presence of auxin, while the levels of the SRPs were decreased by auxin and increased by abscisic acid. Short and long-term effects of 2,4-D and abscisic acid on the synthesis of specific polypeptides were observed, allowing a discrimination between the direct and indirect effect of both hormones on the development of cell senescence.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

Summary Embryogenic callus was induced from primary leaves of Vigna unguiculata (L.) Walp. in MS medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D). Greenish-white, friable embryogenic calluses were used to establish suspension cultures. A shaking speed of 90 rpm and 0.4 ml packed cell volume per 25 ml medium were found to be optimal for maintaining suspension cultures. Globular, heart-shaped and torpedo-shaped embryos were developed in suspension culture containing 4.52 μM 2,4-D. Maturation of cotyledonary-stage somatic embryos was achieved on 0.05 μM 2,4-D, 5 μM abscisic acid and 3% mannitol. Twenty-two percent of the embryos were converted into plants and survived; survival in the field was 8–10%.     

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells: II. Influence of 2,4-D Metabolism and Intracellular pH on the Control of Cell Division by Intracellular 2,4-D Concentration

The utilization of 2,4-dichlorophenoxyacetic acid (2,4-D) molecules by Acer pseudoplatanus cells is governed mainly by a glucosylation process. Evidence that 2,4-D glucoside molecules are biologically inactive is presented. 2,3,5-Triiodobenzoic acid (TIBA), by inhibiting 2,4-D glucosylation, has a sparing effect on 2,4-D molecules; thus TIBA treatments increase growth yield (expressed as the ratio of the maximum number of cells produced to the initial concentration of 2,4-D in the culture medium).     

Purification and immunohistochemical detection of an embryogenic cell protein in carrot

An embryogenic cell protein from carrot (Daucus carota L.), designated ECP31 for embryogenic cell protein and with a relative mass of 31,000, was purified by sequential column chromatographies. Its apparent relative mass was estimated to be 120,000 by gel filtration. Immunoblotting and immunohistochemical studies showed that ECP31 was preferentially localized in the peripheral cells of clusters of embryogenic cells in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) and disappeared during the course of somatic embryogenesis in the absence of 2,4-D. ECP31 began to accumulate on the 33rd day after initiation of cultures of hypocotyl segments on Murashige-Skoog medium with 2,4-D, when callus began to appear on the segments. In dry seeds, lower amounts of ECP31 were located throughout the entire zygotic embryos but not in endosperm. ECP31 was also detected in provascular tissue of malformed somatic embryos.     

Auxin requirements of sycamore cells in suspension culture

Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this `auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.

There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10⁶ cells) on day 2 followed by a decline to 45 nanograms per 10⁶ cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10⁶ cells) rises slowly to a peak of 1.4 nanograms per 10⁶ cells by day 9 then decreases rapidly to 0.2 nanograms per 10⁶ cells by day 15 (early declining phase) and 0.08 nanograms per 10⁶ cells by day 23 (early stationary phase).

     

Effect of 2,4-dichlorophenoxyacetic Acid on glucosylation of scopoletin to scopolin in tobacco tissue culture

2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated the formation of scopoletin and scopolin in tobacco (Nicotiana tabacum L. `Bright Yellow') cell culture. It especially stimulated the uptake of scopoletin from culture medium into the cells and the glucosylation of scopoletin to its monoglucoside, scopolin. This phenomenon is peculiar to 2,4-D, in contrast to other plant hormones. 2,4-D (1 μg/ml) stimulated the glucosylation of scopoletin to scopolin by enhancing UDP-glucose:scopoletin glucosyltransferase (SGTase) activity. The enhancement of SGTase activity caused by treatment with 2,4-D was observed when the syntheses of RNA and protein were inhibited by either actinomycin-D and/or cycloheximide. However, the stimulatory effect of 2,4-D was inhibited by treatment with dinitrophenol. Furthermore, SGTase with or without treatment by 2,4-D in vivo for 24 hours, was isolated from cultured tobacco cells. The enzymes were purified about 200-fold by precipitation with (NH₄)₂SO₄ and chromatography with Sephadex G-100, DEAE-cellulose, and hydroxyapatite. The specific activity of 2,4-D-treated SGTase was 10 times higher than that of untreated SGTase even in the purified fraction, which showed one protein band under electrophoresis. These results suggest that the enhancement of SGTase activity by 2,4-D is due to the energy-dependent activation of the enzyme already present, but not due to the de novo synthesis of the enzyme.     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.     

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.     

High-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l^?1 of α-naphthaleneacetic acid (NAA) and 1 mg l^?1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.     

Plantlet formation from cultured inflorescences of Dactylis glomerata L.

The objective of this study was to demonstrate plantlet formation from cultured mature inflorescences of field-grown orchardgrass (Dactylis glomerata L.) plants. Whole tillers were collected and maintained in the dark at 4°C for either 19 or 25 days before panicle sections were plated on a Linsmaier and Skoog (LS) or Schenk and Hildebrandt (SH) agar medium containing 2,4-D. Generally, better results for both cell proliferation and plantlet formation were obtained with 1) large explants (many florets) on 9.1 μM 2,4-D compared to small explants (few florets) on 1.0 μM 2,4-D, 2) SH rather than LS medium and 3) when tillers were pretreated at 4°C for 25 days rather than 19 days. Chromosome counts of basal leaf cells in 94 regenerated plants showed that no plants possessed the gametic chromosome number of n=14.     

Establishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells

A system of synchronous cell division was established by starvation of auxin and its readdition to suspension cultures of cells of Catharanthus roseus L. cv. Little-Pinky. When cells in the stationary phase were transferred to fresh medium free of 2,4-dichlorophenoxyacetic acid (2,4-D), cells were arrested preferentially at the G₁ phase. After cells had been cultured for 2 days in medium without 2,4-D, readdition of 2,4-D induced the synchronous division of cells. In this system, 70–80% of cells divided synchronously within 3 to 4h, and the mitotic index increased sharply in parallel with the increase in cell number. Active synthesis of DNA was demonstrated by measurements of incorporation of [³H]-thymidine into the DNA fraction. The induction of cell division by the addition of 2,4-D was inhibited by treating cells with analogues of auxin, such as 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxyisobutyric acid.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - IAA indole-3-acetic acid - MS Murashige & Skoog - NAA -naphthalenacetic acid - PCIB p-chlorophenoxyisobutyric acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid     

Stress-induced somatic embryogenesis in vegetative tissues of Arabidopsis thaliana

Somatic embryogenesis is an obvious experimental evidence of totipotency, and is used as a model system for studying the mechanisms of de-differentiation and re-differentiation of plant cells. Although Arabidopsis is widely used as a model plant for genetic and molecular biological studies, there is no available tissue culture system for inducing somatic embryogenesis from somatic cells in this plant. We established a new tissue culture system using stress treatment to induce somatic embryogenesis in Arabidopsis. In this system, stress treatment induced formation of somatic embryos from shoot-apical-tip and floral-bud explants. The somatic embryos grew into young plantlets with normal morphology, including cotyledons, hypocotyls, and roots, and some embryo-specific genes (ABI3 and FUS3) were expressed in these embryos. Several stresses (osmotic, heavy metal ion, and dehydration stress) induced somatic embryogenesis, but the optimum stress treatment differed between different stressors. When we used mannitol to cause osmotic stress, the optimal conditions for somatic embryogenesis were 6-9 h of culture on solid B5 medium containing 0.7 m mannitol, after which the explants were transferred to B5 medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 microm), but no mannitol. Using this tissue culture system, we induced somatic embryogenesis in three major ecotypes of Arabidopsis thaliana-Ws, Col, and Ler.     

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.     

Effect of 2,4-dichlorophenoxyacetic acid on the activity of o-methyltransferase in carrot cell culture

The activity of caffeic acid-O-methyltransferase (OMT) in carrot cells was greatly affected by the amount of 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented to the culture medium. The OMT fraction was purified by (NH₄)₂SO₄ followed by ultrafiltration and gel filtration or DEAE-Sephadex chromatography after cells were cultured in the medium containing [2-¹⁴C]-2,4-D. Thus, this purified fraction revealed high OMT activity and was still radioactive. The OMT activity was about eight-fold higher (or more) in cells cultured at 0.05 ppm 2,4-D than in those at 1.0 ppm 2,4-D. The ratio of radioactivity to OMT activity was about four-fold higher in cells cultured at 1.0 ppm 2,4-D than those at 0.05 ppm 2,4-D. On the other hand, the OMT fraction was separated into two radioactive protein fractions by DEAE-Sephadex chromatography. The radioactive fractions became Et₂O-soluble after HCl hydrolysis, but not after salt-urea treatment. From these results, it was concluded that 2,4-D is covalently bound to proteins in the OMT fraction. Such 2,4-D protein conjugates may play a role in the regulation of OMT activity.