Inhibition of ethylene action prevents root penetration through compressed media in tomato (Lycopersicon esculentum) seedlings

In the genus Prunus , so far, somatic embryogenesis has not been reported either from cell suspensions or from their protoplast-derived cells. Rhizogenic cell suspensions of Prunus avium L., initiated from adventitious roots developed from cotyledon-derived callus of mature zygotic embryos, have been subcultured for more than one year without losing their morphogenic potential. A yield of 8 × 10⁵ protoplasts ml⁻¹ of packed cells with a viability of 98% has been routinely obtained. Optimum cell division frequency (around 2.5% at day 10 and 4–6% at day 15) occurs in agarose lenses, with Murashige and Skoog (1962. Physiol. Plant 15: 476–497)-based medium supplemented with 5 μ M naphthalene acetic acid, 1 μ M benzyladenine and 0.25 μ M zeatin. Colony formation has been achieved after 35 days with a plating efficiency of 3–4%. Cell suspensions have been initiated from protoplast-derived callus. While the older cell cultures express a rhizogenic response, the younger ones contain early stages of somatic embryo development. Ultrastructural examination confirms the polarization of these structures.     

Callus formation from cotyledon protoplasts of Pinus oocarpa and Pinus patula

Protoplasts were isolated from cotyledons of 11-day-old seedlings of Pinus oocarpa and P. patula ssp. tecunumanii . The best enzyme combination was Cellulase R10 + Pectolyase Y-23, associated with bovine serum albumin. When cultured at a low density [1.25 × 10³ to 5 × 10³ protoplast (ml)⁻¹] in a liquid medium, the cells divided. The medium contained glutamine and casein hydrolysate as nitrogen sources, and glucose as osmoticum. Rate of division was increased by supplementing the medium with l -ornithine, putrescine and spermidine. However, the rate remained low, with an absolute division frequency of ca 1%. Dilution allowed colony proliferation and fragmentation, leading to the formation of numerous microcalli that could be transferred to various solid media for further growth.     

The effect of a calcium-channel antagonist, nifedipine and agonist, Bay K-8644, on the phytochrome-controlled swelling of etiolated wheat protoplasts

The effect of external Ca²⁺ and Ca²⁺-channel modulators on the phytochrome-controlled swelling of etiolated wheat ( Triticum aestivum L. cv. Arminda) mesophyll protoplasts has been studied. The red light (R)-stimulated swelling of the protoplasts requires Ca²⁺ in the surrounding medium and maximum response was observed in a medium containing I m M CaCI₂. Far-red light (FR) irradiation of protoplasts in the presence or absence of Ca²⁺ does not influence the protoplast volume. The Ca²⁺-channel antagonist nifedipine prevents R-induced protoplast swelling at very low concentrations (0.1 μ M ). The Ca²⁺ -channel agonist Bay K-8644 stimulates the swelling of protoplasts incubated in darkness or irradiated with FR. Action of nifedipine depends on whether it is applied before or after the R pulse. The results are compatible with the hypothesis that phytochrome controls the activity of dihydropyridine-sensitive L-type Ca²⁺ channels.     

Characterisation of Simple In Vitro Cultures of Striga hermonthica Suitable for Metabolic Studies

Abstract: The aim of this study is to develop simplified models for standardised screenings of xenobiotics, especially targeted against mannitol production, to control the harmful parasite, S. hermonthica. Chlorophyllous protoplasts and calli were produced from the young leaves of the parasite. Best yield from protoplast isolation was obtained when leaf segments were incubated at 30 °C, in the light, under shaking in an enzyme cocktail containing 2 % cellulase Onozuka R10, 0.1 % Pectolyase and sorbitol 1 M as the osmoticum. Oxygen exchange measurements, as well as labelling experiments with ¹⁴C-bicarbonate, emphasised a significant decrease in photosynthetic capacity of protoplasts, mannitol remaining, however, a major primary product of photosynthesis. Initiation of cell cultures was unsuccessful and instability of protoplasts prevents their standardised utilisation for herbicide screening. In contrast, globular calli produced first on MS medium containing 0.5 mg L^-1 NAA, 2.5 mg L^-1 BAP and 2 % sucrose were stable for two years, after monthly subculturing on fresh medium. Sucrose substitution by mannose in the medium did not change kinetic growth and stability. Potential autotrophy was lost for calli by increasing exogenous sugar level. Biochemical analyses and labelling experiments with ¹⁴C-bicarbonate or ¹⁴C-sucrose or -mannose showed that carbon partitioning is modified in calli, in comparison with young leaves or protoplasts, sucrose or mannose accumulation being favoured in sucrose- or mannose-fed calli, respectively. However, carbon flow towards mannitol was more marked in calli growing on high mannose medium. Stability and preservation of an active mannitol biosynthetic pathway allows planning of xenobiotic assays with calli as a simplified model for Striga hermonthica.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Effects of nitrogen source, calcium concentration and osmotic stress on protoplasts and protoplast-derived cell cultures of Pinus Pinaster cotyledons

The experiments described emphasize the effects of several factors crucial to the maintenance of cell divisions leading to increased cell numbers in suspension and colony formation from cotyledon protoplasts of Pinus Pinaster Ait. Osmotic potential of the incubation and culture media are critical. Reducing the osmolality from 680 mOsm kg H₂O⁻¹ during protoplast isolation to 610 mOsm kg H₂O⁻¹ during washing and culture was essential to achieve a high frequency of cell division. Survival of the cells beyond 3 weeks of culture occurs only if the calcium concentration is decreased from 5.6 m M to 1.5 m M . Glutamine as sole source of nitrogen shortens the lag phase of response of the protoplasts and increases their plating efficiency. After 6 weeks of culture, a combination of low osmolality (225 mOsm kg H₂O⁻¹) and high level of glutamine (40 m M ) is a prerequisite for obtaining actively growing cell suspensions.     

Microcallus formation from Hevea brasiliensis protoplasts isolated from embryogenic callus

Conditions for successful culture of rubber tree (Hevea brasiliensis) protoplasts were investigated. Protoplasts, derived from embryogenic callus, regenerated cell walls then underwent division when embedded in alginate and cultivated on a modified Murashige and Sook medium (9 M 2,4-dichlorophenoxyacetic acid, 0.6 M glucose, 0.93 M kinetin, NH ₄ ⁺ reduced by half) in the presence of nurse cells (tobacco feeder cell layer). The presence of nurse cells was essential to maintain viability and sustain protoplast division. Several parameters which influenced the plating efficiency were analysed, such as the density of feeder cells and the duration of contact of the feeder layer.Abbreviations BSA Bovine Serum Albumin - CPW Cell and Protoplast Washing medium (Frearson et al. 1973) - 2,4-D 2,4-dichlorophenoxyacetic acid - 3,4-D 3,4-dichlorophenoxy-acetic acid - PDA Fluorescein diacetate - FW Fresh weight - KIN Kinetin - MES [2N-morpholino] ethane sulfonic acid - MS medium Murashige and Skoog (1962) medium - PE plating efficiency - SAB South American Leaf Blight - WPM] Woody Plant Medium (Russel and Mc Cown 1986)     

Biological activity of monomeric Sendai virus HN glycoprotein

Abstract We have developed a transformation system for Streptomyces wadayamensis , a cephamycin C producer. 1−5 × 10⁹ protoplasts can be obtained when late logarithmic phase cultures of this microorganisms are incubated with 10 mg/ml of lysozyme. Polyethylene glycol-Ca²⁺-mediated transformation of these protoplasts yielded 10⁶ transformants per μg of pIJ702 or pIJ365 DNA.     

Isolation and culture of suspension-derived protoplasts ofBeta vulgaris L.

Sugar beet protoplasts (Beta vulgaris L.) were isolated from hypocotyl-derived suspension cells and cultured on modified Murashige and Skoog medium supplemented with 5 μM naphthaleneacetic acid (NAA) and 2 μM 6-benzyl-aminopurine (BAP). Protoplasts were plated at a density 1.0–1.5×10⁵ cm⁻³ and incubated in either liquid medium or in medium solidified by 1.2% agarose, at 25°C in the dark. Comparison of two methods of culture unequivocally showed the second to be superior. Immobilizing the protoplast in agarose proved to be essential for obtaining sustained protoplast division and reproducible colony formation. The plating efficiency after two weeks of culture, expressed as the percentage of protoplasts which developed to form colonies, reached 40%. Subsequent subcultures of protoplast-derived callus to regeneration media with different concentrations of BAP (5 μM, 10 μM, 20 μM, 30 μM) resulted in very good callus proliferation at the three lowest concentrations, although organogenesis was not achieved.     

Division of guard cell protoplasts of Nicotiana glauca (Graham) in liquid cultures

Abstract. Guard cells are uniquely differentiated to transduce signals into the metabolic and ion transport processes that result in turgor-driven stomatal movements. We tested the hypothesis that these highly specialized cells are terminally differentiated. Guard cell protoplasts were isolated from abaxial epidermal tissue of leaves of Nicotiana glauca (Graham) and cultured in a medium designed for culturing mesophyll protoplasts of Nicotiana tabacum. Protoplasts were incubated at densities of 2–5 × 10¹¹ cells m⁻³ in eight-well microchamber slides under 50μmol m⁻² s⁻¹ of photons of continuous fluorescent light at 25°C. When the medium was modified by the addition of 100mol m⁻³ of sucrose and by buffering with 10mol m⁻³ of MES buffer at pH 6.1, cell division began within 96h of the time the culture was initiated. After 9d of culture, 80% of surviving cells had synthesized new cell walls, had dedifferentiated, and were dividing to form small colonies. Callus tissue was visible after 4–5 weeks. We conclude that guard cells of Nicotiana glauca are not terminally differentiated, and that guard cell protoplasts of this species have the capacity to grow, synthesize cell walls and divide.     

High yield isolation of mesophyll protoplasts from wheat, barley and rye

Efficient procedures are described for high-yield isolation of mesophyll protoplasts from spring wheat ( Triticum aestivum L. cv. Glenlea), winter wheat ( Triticum aestivum L. cv. Frederick), barley ( Hordeum vulgare L. cv. Bruce) and rye ( Secale cereale L. cv. Puma). Factors such as plant age, composition of the incubation medium during isolation, purification procedures and culture medium affect protoplast yield, viability and metabolic competence, as measured by light-dependent CO₂ fixation. Optimal osmolarity of the isolation medium was equivalent to 1.8 times that measured in the leaves of all plant material used. The presence of 2 m M ascorbic acid in the preincubation and isolation medium increased the yield by 50% and conserved viability and metabolic competence. The protoplasts were stable for up to 48 h without loss of either viability or of original activity of CO₂ fixation, which was in the order of 100 μmol CO₂ (mg chl)⁻¹h⁻¹.
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided.     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

Influence of phytohormones and other effectors on proton extrusion by isolated protoplasts from rape leaves

The roles of phytohormones and fusicoccin in H⁺ extrusion by isolated protoplasts from rape leaves ( Brassica napus L. cv. Belinda) were investigated and compared to results obtained with leaf segments of the same plants. Net H⁺ release by protoplasts, which was at least partly due to ATPase activity, was enhanced by 10 μ M indole-3-acetic acid and reduced by 20 μ M abscisic acid, whereas fusicoccin (10 μ M ), brassinosteroid (3 μ M ), kinetin (20 μ M ) and gibberellic acid (10 μ M ) had no effect. Hormone effects and H⁺ release were not detectable with leaf segments from the same plants. However, using field-grown plants, indole-3-acetic acid and especially fusicoccin stimulated the acidification of the external medium by leaf segments. Hormonecontrolled H⁺ release by leaf cells is interpreted as the first step in acid-triggered and turgor-regulated cell growth.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

Transformation of Streptococcus bovis protoplasts by plasmid DNA

M. MAREKOVÁ, V. KMET' AND P. JAVORSKÝ. 1996. The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca²⁺ and Mg²⁺ ions (2.5 mmol l^-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 times 10³ transformants per μg of pNZ12 and 2.4 times 10² per μg of pJK108, respectively.     

Arginine utilization by Mycoplasma fermentans is not regulated by glucose metabolism: A 13C-NMR study

Abstract Electrofusion of protoplasts of two mutant strains of Hansenula polymorpha resulted in high fusion and hybrid yields when the calcium ions present in the conventional fusion medium replaced by zinc ions. The optimal fusion conditions were an alignment field of 0.4 kV cm⁻¹ strength and 2 MHz frequency for 30 s, followed by two consecutive pulses of 12 kV cm⁻¹ strength and 15 μs duration. With 0.05–0.1 mM zinc ions in the fusion medium an average clone number of 10⁴–10⁵ clones per 10⁸ input cells was reached. The presence of about 0.6 mM magnesium ions in the zinc fusion medium was essential.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

Rhizogenesis in callus from Conference pear (Pyrus communis L.) protoplasts

Large numbers (ca 6×10⁶ protoplasts/g f.wt) of viable (80%) protoplasts were isolated from embryo-callus tissues of Conference pear using an enzyme mixture which contained 2.0% (w/v) Meicelase, 2.0% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. A medium based on ammonium-free MS salts and supplemented with 2.0 mg/l NAA, 0.5 mg/l BAP and 9% (w/v) mannitol supported protoplast division and the proliferation of multicellular colonies. Colonies were taken to the callus stage on a medium which contained MS salts plus 0.1 mg/l 2,4-D and 0.1 mg/l BAP. Roots were regenerated from these protoplastderived calli on MS medium with 0.1 mg/l NAA, 5.0 mg/l BAP and 50 mg/l casein hydrolysate.Abbreviations BAP 6-benzylaminopurine - CPW13M CPW salts medium [15] with 13% (w/v) mannitol - FDA fluorescein diacetate, f. wt-fresh weight - MS Murashige and Skoog [14] - NAA -naphthaleneacetic acid - PE plating efficiency (%) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Regulation of lateral root formation in lettuce (Lactuca sativa) seedling roots: Interacting effects of α-naphthaleneacetic acid and kinetin

Growth substances, α-naphthaleneacetic (NAA) and kinetin, had an important role in the regulation of lateral root (LR) formation in lettuce ( Lactuca sativa L. cv. Grand Rapids) seedling roots. NAA (10^-5 M ) was a potent stimulator of LR initiation and caused a 600% increase in the number of lateral root primordia (LRP) compared to untreated roots. NAA was required for only the first 20 h of the 72 h treatment period for maximum stimulation of LRP initiation. Kinetin (2 × 10^-5 M ) effectively prevented the spontaneous formation of LRP and inhibited the NAA-stimulated production of LRP. Kinetin inhibition was maximal during the first 20 h of NAA treatment and this effect was not overcome by subsequent supply of NAA. Also, lettuce roots were most sensitive to kinetin at 20 h of NAA treatment, when the first signs of cell division were observed in the pericycle.