Production of a constitutive,extracellular levansucrase fromErwinia herbicola NRRL B-1678

Summary Levansucrase (E.C. 2.4.1.10), which has long been known to be produced intracellularly byErwinia herbicola, was found in relatively high concentrations in the extracellular culture fluid of bacteria grown in various media. Sucrose was not required for enzyme secretion. A medium consisting of corn steep liquor and sorbitol or mannitol gave the highest yields of enzyme.The mention of firm or trade names does not imply endorsement by the USDA or recommendation over other firms or similar products not mentioned.     

Permeabilization of Cinchona ledgeriana cells by dimethylsulphoxide. effects on alkaloid release and long-term membrane integrity

Dimethyl sulphoxide (DMSO) has been used to permeabilize cells of Cinchona ledgeriana in suspension culture and promote the release of intracellular alkaloids. 5–6% v/v is required before any release is seen, and greater than 20% DMSO is required for full release. Even at these high levels of DMSO release is slow, taking in excess of seven hours to reach completion. Conditions which produce significant release of alkaloids have a deleterious effect on cells. Many of the membranes permeabilized did not recover their ability to selectively exclude compounds such as mannitol when the DMSO was removed. It is concluded that DMSO is not a suitable material for inducing alkaloid release in any biotechnological exploitation of alkaloid production by C. ledgeriana.Abbreviations DMSO Dimethyl sulphoxide - 2,4D 2,4-Dichlorophenoxyacetic acid     

Production and regeneration of protoplasts of Pisolithus tinctorius

Summary The production of protoplasts of the ectomycorrhizal fungus Pisolithus tinctorius, isolate Pt 571, was improved using young mycelium treated with Novozym-234 dissolved in 0.5M mannitol in the proportion 20:1:0.15 (mg mycelium: mg enzyme: ml mannitol) and incubated at 25°C for 4 hours. Plating the protoplasts on the surface of solid medium containing 0.5M mannitol increased the regeneration rate.     

Transformation of Medicago arborea L. with an Agrobacterium rhizogenes binary vector carrying the hygromycin resistance gene

Plants of Medicago arborea have been infected with Agrobacterium rhizogenes strain LBA9402 harbouring the plasmids Ri 1855 and AGS125 carrying a gene conferring resistance to the antibiotic hygromycin. About 7056 of the hairy roots showed callus formation on hygromycin-supplemented medium. Regeneration took place on antibiotic free medium only. Plantlets suitable for transfer to soil were obtained after the manual removal of most of the leaves. Plant morphology showed the usual alterations induced by the Ri plasmid; moreover, two years after soil-transfer, transformants have not flowered. Molecular analysis indicates the presence of T-DNA from both pAGS 125 and p1855. The expression of the hygromycin phosphotransferase gene allowed callus and protoplasts of transformed plants to grow on media supplemented with the antibiotic. This trait will be utilized as a marker in protoplast fusion between Medicago arborea and Medicago sativa (alfalfa).Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - kin kinetin - GA3 Gibberellic acid - IAA Indole-3-acetic acid - HPT hygromycin phosphotransferase - NOS nopaline synthase - MS Murashige and Skoog (1962) - B5 Gamborg et al. (1968) - B5hy B5 supplemented with 20 mg 1-1 of hygromycin - YMB yeast mannitol broth     

In situ detection of transposition of the maize controlling element (Ac) in transgenic soybean tissues

The development of a transposon mutagenesis system in soybean would aid in the isolation of unknown genes. The maize controlling element (Ac) has, therefore, been introduced into the soybean (Glycine max (L.) Merr.) genome byAgrobacterium-mediated transformation.Ac was inserted into the untranslated leader region of the bacterial ß-glucuronidase gene (GUS) such that the excision ofAc resulted in restoration of the GUS gene activity. Excision events of theAc element were monitored by detecting blue cells or sectors in transgenic soybean tissues. Using the GUS gene assay and with hybridization data, we have demonstrated that theAc element transposes in transgenic soybean calli, leaves, stems, and roots.     

Protein synthesis in a maize callus exposed to NaCl and mannitol

A maize (Zea mays, L) callus was exposed to media containing different levels of NaCl (0 to 3%) and mannitol (0 to 18.2%) for a period of 4 weeks, and the changes in growth and protein synthesis were determined. Cells are able to tolerate and grow in NaCl up to 1% (0.17 M) or mannitol up to 9.1% (0.5 M), but the relative overall growth rates are about 1/6 and 1/8 of the control, respectively. Protein synthesis, as assessed by pulselabeling of the cells with ³⁵S-methionine after exposure to the stress reagents at various times of incubation, suggests that the relative rates of amino acid uptake and its incorporation into proteins are inhibited as early as 4 h after exposure, and the extent of inhibition does not increase appreciably until after 1 week. Severe inhibition of uptake and protein synthesis results from prolonged exposures at growth-inhibitory concentrations of NaCl and mannitol. In general, the overall mean inhibition of cellular uptake and protein synthesis in the first 2-week period are approximately 50% and 35% for the NaCl (1%) and mannitol (7.3 %) treatments, respectively. No detectable differences are apparent in the abundant, steady state protein population as revealed by SDS-PAGE and on staining with Coomassie blue or silver, but random losses of individual proteins occur after 2 weeks at 2% and 3% NaCl and at 18.2% mannitol. Of the newly-synthesized proteins, discernible changes are found in 7 and 4 polypeptides in NaCl and mannitol treatments, respectively. Apparently three new proteins (74 kd, 28.5 kd, and 26.2 kd) are induced de novo under both treatments. Other proteins (39.5 kd, 39.0 kd, 30 kd, and 16.5 kd) show an increased or decreased level of synthesis. NaCl levels above 0.5% or mannitol levels above 3.6% do not after the pattern of newly-synthesized proteins. This altered expression of newly-made proteins in the maize callus occurs only after a week of exposure to salinity or osmotic stress and coincides with the cell growth phase.     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

A new protocol for isolation of mitochondrial DNA from cotton seedlings

A procedure to isolate mtDNA from cotton seedlings G. hirsutum and G. barbadense has been developed. The new protocol allows for the isolation of cotton mtDNA of high purity, yield and digestibility by restriction endonucleases. The success of the protocol is based on critical adjustments in the ionic strength of the homogenizing medium and washing buffer, the speed of grinding during homogenization, and the methods used for lysis of the mitochondria.     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

A new bioassay system for screening high berberine-producing cell colonies of Thalictrum minus

A new method of estimating the amount of berberine released from minute cell colonies of Thalictrum minus has been devised to facilitate the selection of high berberine-producing cell lines. In this system, cell aggregates obtained from a cell suspension culture are grown on small pieces of an agar culture medium and the concentration of berberine which has been released from the cells into the agar piece is assayed by the antibacterial activity against Bacillus cereus MT2026. Screening of 1000 cell colonies by the agar piece method has resulted in the isolation of four, high berberine-producing cell lines, although they have been found to be more or less unstable with respect to the biosynthetic capability during successive subcultures.     

A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts

Protoplasts isolated from suspension cultures of atrazine resistant black nightshade (Solanum nigrum L.) a weed biotype, were enucleated by centrifugation through a stepwise mannitol/sucrose gradient. Two cytoplast, enucleated subprotoplast, bands were routinely formed: one, a minor band at the 6.4%/18.2% mannitol border containing highly vacuolate cytoplasts with 95%+ enucleation; secondly a major cytoplast band at the 18.2% mannitol/33% sucrose border containing 90%+ enucleated protoplasts in quantities up to 4 million per 50 ml gradient tube. Efficient production of cytoplasts depended on the subculture procedures used for the cell suspensions. Optimal cytoplast yield (44%) occurred for protoplasts isolated three days after subculture. The vigor of the donor suspension cultures as visually monitored had to be controlled in order to obtain consistently high enucleation percentages.Abbreviations CPW Cell and Protoplast Wash Solution - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - FDA Fluorescein diacetate - MS Murashige and Skoog medium (1962) - UM Uchimiya and Murashige medium (1976)     

Plant regeneration from stem cortex protoplasts of a tomato hybrid

Longitudinal sections containing cortical cells taken from stem internodes of a hybrid betweenLycopersicon esculentum andSolanum lycopersicoides were used as tissue sources for enzymatic protoplast isolation. Greenhouse and growth room-grown plants 4–8 weeks after rooting could be used as sources of donor tissue. Protoplasts from these tissues divided within 2–4 days of culture and numerous microcalli formed within 30 days. The shoot regeneration frequency of protoplast-derived calli was in the order of 60%. More than 100 regenerated plants which appear phenotypically normal have been established in soil.Abbreviations NAA 1 naphthaleneacetic acid - 2,4-D 2-4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA 6 benzylaminopurine - FDA fluorescein diacetate     

Cell regeneration and sustained division of protoplasts from cotton (Gossypium hirsutum L.)

Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbiochem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K₃ media of Kao et al., 1974 with 0.9 M BAP, 5 M IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5×10⁵ protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed.Abbreviations IAA indoleacetic acid - BAP 6-benzyl-adenine Arizona Experimental Station Publication No. 4373     

Production of large number of doubled haploid plants from barley anthers pretreated with high concentrations of mannitol

Pretreatment with increasing concentrations of mannitol, from 0.3 to 0.7 M, was used to induce stress in cultured anthers of barley (Hordeum vulgare L.). Three cultivars with varying degrees of androgenetic ability were studied. A positive linear relationship was found between concentration of mannitol in the pretreatment medium and the number of regenerated green doubled haploid plants in all the cultivars. The pretreatment also resulted in an increasing proportion of embryos to dividing microspores, and in green to albino plantlets. The optimum length of the pretreatment seemed to be genotype dependent. When Ficoll was used as an alternative stress agent a differential genotype response was observed.Abbreviations BAP N⁶ benzyl-aminopurine - IAA indol acetic acid     

Production of ergot alkaloids by Aspergillus fumigatus (Fresenius)

Summary Nutritional requirements for the production of ergot alkaloids were studied with Aspergillus fumigatus under submerged conditions of fermentation, in a chemically defined medium. Glucose in combination with mannitol and triammonium citrate were found to be the best sources of carbon and nitrogen for the production of alkaloids. Carbon to nitrogen ratio of 4.16 : 1 was found optimum. Phosphate at elevated concentration inhibited alkaloid production.     

Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Callus culture as substrate for the production of specific cell wall degrading enzymes for derived protoplast isolation

Callus culture of spruce (Picea excelsa LINK) appears to be a suitable substrate for the fungusTrichoderma reesei to produce an efficient extracellular lytic system for protoplast isolation. In comparison with Onozuka R-10 cellulase, a yield of protoplasts from the spruce callus 2·5 higher was obtained. Another testea commercial cellulase DK was less efficient. The addition of Macerozyme R–10 significantly enhanced release of protoplasts within all tested enzyme preparations. No difference in the viability of protoplasts has been observed.     

A rapid method for the isolation of plasmid DNA from bacteria

Summary A rapid method is described for the isolation of plasmid DNA from Escherichia coli and Pseudomonas putida. The effect of heating the cell preparation during plasmid extraction is discussed in relationship to the final plasmid yield.     

Accumulation of anthocyanins enhanced by a high osmotic potential in grape (Vitis vinifera L.) cell suspensions

A cell suspension of grape, Vitis vinifera L. cv Gamay Fréaux, was grown under different conditions of water stress (high external osmotic potential) induced by an increase of sucrose concentration or by the addition of mannitol to the culture medium. Best growth (cell density) was achieved in the low osmotic potential medium. Increasing the osmotic potential of the medium from –0.5 MPa to –0.9 MPa medium resulted in a significant increase in accumulation of anthocyanins in pigmented cells. Regulation of the osmotic potential of culture medium may be useful in controlling anthocyanin production.