The selection of lactose-utiljzing,polysaccharide-producing strains ofXanthomonas campestris

Summary A procedure was developed to discriminate between singleXanthomonas campestris colony isolates which differed in growth form on solid media and in viscosity production in liquid media. The method was used to assess degree of variability between colonies and between successive generations of isolates chosen for industrial application.     

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     

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)     

Cryopreservation of immature embryos of Theobroma cacao

Immature, white zygotic embryos of Theobroma cacao L. (cacao) retained the ability to produce callus and to undergo somatic embryogenesis after slow hydrated freezing and desiccated fast freezing in liquid nitrogen. The highest rate of somatic embryogenesis occurred in embryos which were precultured on a medium containing 3% sucrose, frozen slowly with cryoprotectants before exposure to liquid nitrogen, and recovered on a medium containing 3 mg/liter NAA. Embryos precultured on media containing sucrose increasing to 21% had a higher rate of survival but were less embryogenic after freezing. These results suggest that immature embryos might be used for long-term germplasm storage of T. cacao germplasm.     

A parenchymatic medium solidifier for plant in vitro culture

A new material for the solidification of liquid culture media was prepared from plant parenchyma tissues by mechanical subdivision, solute extration and dessication from ethanol. It is suitable for in vitro culture and propagation of callus as well as shoot tip cultures. The following plant materials have been grown by means of the new medium solidifier: shoot cultures of Betula pendula Roth, Gerbera jamesonii H. Bolus ex Hook and Floribunda rose "Triumph", callus tissues of Daucus carota L. and Chenopodium album L. The new solidifying material has special advantages over agar for application in the rooting phase of in vitro propagation.Abbrevations PMS parenchymatic medium solidifier - MS Murashige and Scoog's medium - IAA Indole-3-acetic acid - B biotin - K kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - ch caseine hydrolysate     

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.     

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.     

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.     

Organogenesis in cell cultures of leafy spurge (Euphorbiaceae) accessions from Europe and North America

Plants were regenerated from leafy spurge (Euphorbia esula L.) cell suspensions obtained from stem callus. A North Dakota accession was highly regenerable, but two accessions from Oregon and Austria formed only a few plantlets. Organogenesis occurred in media without growth regulators, under fluorescent lights (30 to 90 E m^–2 s^–1, 14 h photoperiod). Organogenesis was greatest in larger size clumps subcultured during maximum cell growth into media containing a reduced:oxidized nitrogen ratio of 33:67. Roots formed first and some clumps produced shoots. Organogenic suspension cultures also were initiated from hypocotyl and root segments of germinated seedlings, directly in liquid medium. Plantlets of the North Dakota accession formed in vitro adapted to greenhouse conditions. They were phenotypically similar to the parent plants.     

N-carboxymethylchitosan inhibition of aflatoxin production: Role of zinc

Aqueous Solutions of N-carboxymethylchitosan (NCMC) suppressed both growth and aflatoxin production byAspergillus flavus andA. parasiticus in submerged culture (Adye and Mateles A&M). Test media were amended with various concentrations of zinc (15, 30, 45, 60 uM), and NCMC solution (0.62 uM). After 8 days incubation NCMC-treated cultures showed marked reduction of aflatoxin production and fungal growth. Enhanced levels of zinc did not overcome the NCMC-mediated inhibition of fungal growth or aflatoxin production.     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

Production of D-xylanases by thermophilic fungi using different methods of culture

Summary Seven thermophilic strains of fungi were examined for their ability to produce D-xylanase in Liquid and solid-state fermentations. It was confirmed that the best producers of xylanase, among microorganisms used, wereH. Lanuginosa andS. thermophile in liquid fermentation, andT. aurantiacus andH. lanuginosa in solid-state fermentations. The higher productivity of xylanase, namely 18,72 IU/ml, was obtained in liquid culture ofH. lanuginosa. The pH and temperature optima of enzymes from liquid and solid-state cultures of fungi used were also presented.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Mevalonic acid increases trans-astaxanthin and carotenoid biosynthesis in Phaffia rhodozyma

Summary Mevalonic acid has been tested as enhancer of pigment biosynthesis in wild-type Phaffia rhodozyma. The addition of 0.1% mevalonic acid to the culture media stimulated both trans-astaxanthin and total carotenoids biosynthesis, with average increases by ca 400%.     

Anther culture of some perennial triticeae

Three field grown Agropyron spp. (crested wheatgrasses) and two Thinopyrum spp. (intermediate and tall wheatgrasses) were evaluated for anther culture response. Hormonally modified potato extract and 85D12 media induced pollen embryogenesis. Modified Murashige and Skoog media were tested for their effects on callus proliferation and plantlet regeneration. Callus induction frequency and plantlet production were highest (25.0% and 45.8%, respectively) for Thinopyrum ponticum (2N=70) (tall wheatgrass). One-hundred and nine albino plantlets were produced from T. ponticum Jose both by direct regeneration on 85D12 medium and through a callus phase from potato extract media. This is the first report of plantlet production from anther culture of a Triticeae perennial forage grass. Further experimentation with environmental and cultural conditions may result in the production of green plantlets.Abbreviations MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-ip 2-isopentenyladenosine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid Cooperative investigations of the USDA-Agricultural Experiment Station and the Utah Agricultural Experiment Station, Logan, UT 84322. Approved as Journal Paper No. 3596     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

CMCase production by Spicellum roseum in liquid and solid culture

Summary CMCase was produced by 7 strains of Spicellum roseum in both liquid and wheat bran solid substrate cultures. No growth occurred above 35°C. Maximum enzyme production occurred at 30°C, whereas best enzyme activity occurred at pH 5.0 and 50°C. In liquid cultures of S. roseum, NRRL strains 13103, 13104, and 13106 produced activities of ca. 1.1, 1.5, and 1.5 mg glucose per hr/ml culture supernate at 1 week and 2.9, 1.5, and 2.1, respectively at 3 weeks compared to Trichoderma reesei NRRL 11236 (MCG77), which produced activities of 2.8 and 1.3 at 1 and 3 weeks.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Chitin synthesis inhibitors prevent cyst formation by Entamoeba trophozoites

Cysts of Entamoeba invadens obtained under axenic culture conditions have been reported to be similar to cysts of the human intestinal parasite E histolytica both in morphology and chitin presence in their wails. Mature E. invadens cyst forms, isolated from cultures following discontinuous Percoll gradient sedimentation were resistant (>80%) to detergent treatment. Addition of chitin synthesis inhibitors such as Polyoxin D and Nikkomycin (50 μg/ml) to cultures in encystation media markedly inhibited (>85%) the formation of detergent resistant cysts and prevented the incorporation of radiolabeled chitin precursor N-acetyl[³H]glucosamine. These findings suggest that chitin synthesis inhibitors may serve as drugs which specifically block the life cycle of the Entamoeba parasite.     

Somatic embryogenesis in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Embryonal-suspensor masses from immature embryos from cones of Sitka spruce (Picea sitchensis (Bong.) Carr.) proliferated on a modified Murashige & Skoog medium with N⁶-benzyl-aminopurine, kinetin, 2,4-dichlorophenoxyacetic acid and an organic nitrogen source. The slimy white embryonal-suspensor masses with proembryos were maintained on a solid proliferation medium with reduced amounts of growth regulators. Transfer of embryonal-suspensor masses to a non-woven polyester carrier with liquid maturation media containing ±2-cis-4-trans-abscisic acid and a reduced amount of inositol and organic nitrogen resulted in synchronized embryo formation. Further development was achieved on a medium without ±2-cis-4-trans-abscisic acid and organic nitrogen. Somatic embryos were successfully transferred ex vitrum.Abbreviations ABA ±2-cis-4-trans-abscisic acid - BAP N⁶-benzyl-aminopurine - ESM embryonal-suspensor masses - KIN kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effect of NaCl on lipid content of plasma membranes isolated from roots and cell suspension cultures of the dicot halophyte Kosteletzkya virginica (L.) Presl.

Lipid concent was examined in plasma membrane fractions isolated by discontinuous sucrose density-gradient centrifugation from both salinized and unsalinized roots and cell suspension cultures of Kosteletzkva virginica (L.) Presl., seashore mallow, a halophytic dicot. The distribution of marker enzymes along the gradient indicated that plasma membranes of roots and cell cultures accumulated primarily at the 34%/45% interface. Total sterol and phospholipid content increased significantly in plants and cell suspensions grown on salinized nutrient media. In addition, K. virginica plasma membranes were constitutively rich in sterols, and a high sterol-to-phospholipid ratio was maintained or elevated under saline conditions. These results are discussed in relation to membrane composition as a mechanism involved in the cellularly based salt tolerance of K. virginica.