Plasmid-encoded ropiness production inLactobacillus casei SSP.casei

Summary Genetic determinants of the Muc⁺ character were investigated in two ropy strains,Lactobacillus delbrueckii ssp.bulgaricus 201 andL. casei ssp.casei NCIB 4114, which secrete a large amount of slime in culture media. Plasmid DNA analysis revealed the presence of two plasmids (4.5 and 2.3 Mdal) inL. casei ssp.casei, whileL. delbrueckii ssp.bulgaricus was plasmid free, suggesting a chromosomal location of Muc⁺ character in this strain. Curing experiments carried out onL. casei ssp.casei NCIB 4114 indicated a correlation between the Muc⁺ phenotype and the 4.5 Mdal plasmid.     

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.     

A tissue culture method for rapid clonal propagation of mature trees of Eucalyptus torelliana and Eucalyptus camaldulensis

Multiple shoots were induced from nodal segments of mature Eucalyptus torelliana F. Muell. and E. camaldulensis Dehnh trees on Murashige and Skoog's medium supplemented with Kn, BAP, Cal.Pan and Bio. Incubation in semi-solid media at 15°C with continuous illumination followed by growth in agitated liquid media was essential for shoot induction and development in primary explants of E. camaldulensis. For culture of E. gorelliana, growth in agitated liquid media alone was sufficient. Rooting could be induced in shoot cultures of E. torelliana by treatment with NAA whereas treatment with a mixture of IAA, IBA, IPA and NAA was essential for E. camaldulensis. After auxin treatment, transfer to a charcoal-containing medium was necessary. Rooted plantlets could be successfully transferred to pots and field. By this method it is estimated that about 50,000 plantlets of E. torelliana and 20,000 of E. camaldulensis can be produced, in a year, from a single nodal segment of a mature tree.Abbreviations Kn Kinetin - BAP 6-Benzylaminopurine - Ca.Pan Calcium Pantothenate - Bio Biotin - NAA -Naphthalene acetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid Communication No. 3315     

Solvent production byClostridium pasteurianum in media of high sugar content

Summary The fermentation end products ofClostridium pasteurianum ATCC 6013 are normally acetic and butyric acids. When grown in media of high sugar content however, significant quantities of solvents (acetone, butanol and ethanol) were produced. Solvent production was not stimulated by added acetic and butyric acids, nor was the effect due to a low water activity of the mediumper se.     

Applicability of the PCR technique in the food testing laboratory: Identification of Listeria monocytogenes

A polymerase chain reaction (PCR) targeting Listeria monocytogenes-specific hlyA gene sequences was evaluated as a tool for the identification of presumptive positive colonies isolated on Palcam or Oxford agar during routine analysis of food samples by culture technique. The PCR correctly identified L. monocytogenes isolates from a large number of samples, and provided a significant saving in the time, labour and cost compared to the traditional biochemical tests.     

Induction of somatic embryogenesis in leaf-derived callus of Vicia narbonensis L.

A method for the induction of somatic embryogenesis in callus cultures, using explants from mature leaves of Vicia narbonensis L., is described. Callus developed on a solid medium (Murashige and Skoog 1962), which was supplemented with low concentrations of picloram and benzylaminopurine. Subsequent culture was carried out in different liquid media (culture length four months). The gradual reduction of auxin and cytokinin concentrations, and the addition of glutamine and pyridoxal·HCl were favourable. Somatic embryos appeared on solid media without phytohormones.Abbreviations MS Murashige and Skoog (1962) - 2,4 D dichlorophenoxy acetic acid - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - picloram 4-amino-3,5,6-trichloropicolinic acid - NAA 1-naphthalene acetic acid - p-CPA parachlorophenoxy acetic acid - M1 - M7 media numbers (details in materials and methods)     

Effect of surface active agents on microbial culture

Summary The concentration of several surface active agents required to inhibit the initiation of growth ofAspergillus foetidus in both solid and liquid media was estimated. The culturing in liquid media involved both shake flask and fermenter cultures.     

In vitro culture of pods from annual and perennial Medicago species

Because most interspecific Medicago embryos abort before they can be excised and cultured, our objective was to grow young pods in vitro. Various media were used to grow three-day-old pods of annuals [diploids, M. blancheana Boiss., M. disciformis DC., tetraploid M. scutellata (L.) Mill.] and perennials (diploid M. falcata L., tetraploid M. sativa L.).Few pods of perennial species grew to maturity on media containing modified Hoagland's plus 1% glucose or sucrose with or without 5% potato extract. Increasing sucrose to 6% increased the percentage of M. sativa pods that produced mature seeds. On DM (differentiation medium), the best medium, the percentage of pods producing viable seeds was: M. blancheana (82), M. disciformis (81), M. scutellata (48), M. sativa (63), M. falcata (15). DM plus 1 ppm indoleacetic or gibberellic acid did not enhance seed production.Abbreviations DM Differentiation medium - DMI DM plus 1 ppm indoleacetic acid - DMG DM plus 1 ppm gibberellic acid     

A nucleic acid sequence-based amplification (NASBA) system for Listeria monocytogenes and simple method for detection of amplimers

A NASBA system amplifying specific sequences of the Listeria monocytogenes hlyA gene and an immunoenzymatic assay for the detection of amplimers was developed. The immunoenzymatic assay utilized a simple microtiter plate format and an anti-RNA:DNA hybrid antibody for the detection of NASBA product (predominantly RNA) hybridized to an immobilized DNA probe. This highly sensitive isothermal amplification and detection system was reactive with genomic DNA from various L. monocytogenes isolates but not with other Listeria or non-Listeria species.     

Isolation and selection of phenol - degrading microorganisms from an industrial effluent

Summary Among 12 bacterial and fungal isolates from a phenol-containing effluent, an Acinetobacter sp. and a Fusarium flocciferum strain could be adapted to growth on up to lg/1 of phenol.     

Effect of methionine on the growth and morphogenesis ofSaccharomycopsis fibuligera in defined medium

Summary The growth ofSaccharomycopsis fibuligera on defined media has been investigated. Growth on a minimal medium was stimulated by the addition of methionine but not by the addition of norleucine or other amino acids including cysteine. Growth in the presence of methionine produced pseudomycelium rather than the true mycelium which is produced during growth on complex media A requirement for methionine for growth and pseudomycelium formation inS. fibuligera has been demonstrated.     

Extracellular L-glutaminase production by marine bacteria

Summary Four species of bacteria which includedPseudomonas fluorescens,Vibrio cholerae andVibrio costicola were observed to produce glutaminase both as extracellular and intracellular fractions. Comparatively both the fractions were higher in mineral media supplemented with 1% glutamine than in nutrient broth added with or without glutamine. Extracellular glutaminase production was about 2.6–6.8 times greater than the intracellular production by all the tested strains.     

Interspecific somatic hybridization between lettuce (Lactuca sativa) and wild species L. virosa

Somatic hybrids between cultivated lettuce (Lactuca sativa) and a wild species L. virosa were produced by protoplast electrofusion. Hybrid selection was based on inactivation of L. sativa with 20mM iodoacetamide for 15 min, and the inability of L. virosa protoplasts to divide in the culture conditions used. Protoplasts were cultured in agarose beads in a revised MS media. In all 71 calli were formed and 21 of them differentiated shoots on LS medium containing 0.1mg/l NAA and 0.2mg/l BA. Most regenerated plants exhibited intermediate morphology. These plants were confirmed as hybrids by isoenzyme analysis. The majority of somatic hybrids had 2n=4x=36 chromosomes, and had more vigorous growth than either parent. Hybrids had normal flower morphology, but all were sterile.     

Detection of pectolytic activity andpel homologous sequences inFrankia

Using a cup-plate pectin agar assay, pectolytic activity was detected in nodule filtrates obtained fromAlnus rugosa (DuRoi) Spreng,A. glutinosa (L.) Gaertn andA. crispa (Ait.) Pursh seedlings after infection with twoFrankia strains (ACN1 ^AG , CpI1). Pectolytic activity was also detected in cultures filtrates of the same twoFrankia isolates afterin vitro-cultivation on Qmod pectin liquid medium. When Southern blots of Frankia total DNAs from 3 isolates ofF. alni subsp.Pommerii (ACN1 ^AG , ArI3, and CPX32b) and 3 isolates ofF. elaeagni (EUN1 pec, SCN 10a and TX31e ^HR ) were hybridized withPelBDA probes fromErwinia chrysanthemi, positive signals were found in all 7 Frankiae tested.     

Nature and role of plasmids in Azotobacter chroococcum

Summary Heavy metal, antibiotic resistance and hydrocarbon utilization properties were studied inAzotobactor chroococcum to investigate the nature and role of plasmids in different soil isolates. There is wide prevalence of heavy metal resistance, antibiotic resistance and utilization of hydrocarbons; the heavy metal resistance property seems to be conferred by plasmids.     

Antiplasmodial activity of Artemisia annua plant cell cultures

Extracts of Artemisia annua cultures have been assessed for in vitro activity against the malarial parasite Plasmodium falciparum. Callus and suspension cells and medium were analysed and examined for their activity at different stages of growth and development. Time-course experiments were carried out to investigate the influence of various basal media, plant growth regulators and light on both growth and possible artemisinin production. Two active fractions were obtained but artemisinin was not detected.     

Plant regeneration from hordeum spontaneum and hordeum bulbosum immature embryo derived calli

Callus cultures were initiated from isolated immature embryos of Hordeum spontaneum and Hordeum bulbosum on MS or B5 basal medium supplemented with 2 mg/1 2,4-D. Shoot regeneration occurred on transfer of tissue to media containing 1 mg/1 IAA and 1 mg/1 zeatin. The regenerated shoot buds were rooted on basal medium without hormones. The in vitro regenerated plants were transferred to soil and were grown to fertile mature plants. A low percentage of albino plants was observed among the regenerated plants. No major differences were detected between the two species in respect to their potency to form callus or to the regeneration capacity. The regeneration capacity of calli decreased gradually and ended after 6 months in culture.Abbreviations IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - MS Murashige and Skoog medium     

Biotechniques for improving acid aluminum tolerance in alfalfa

Alfalfa (Medicago sativa L.), cultivars ARC, Regen Y and Saranac were selected in vitro in a recently developed acid/aluminum toxic media. The new media produced higher initiation rates and higher fresh callus weights than those obtainable with the media described by Meredith and Connor for the selection of aluminum resistant variants in Nicotiana plumbaginofolia. Both rescue and direct initiation yielded adequate amounts of healthy callus for the initiation of embryogenesis. The toxic effect of the acid/aluminum media is expressed in both the percent of explants initiating callus and in the fresh-weights obtained during initiation and two subsequent subcultures.     

Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna

Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - LpDH lysopine dehydrogenase