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Summary Q fever is a zoonotic illness which frequently has a non-specific clinical presentation. Cases among deployed US military personnel have been reported in increasing numbers indicating an emerging at-risk occupational group. Banked serum specimens were utilized to estimate seroprevalence and risk factors among military personnel deployed to Iraq. Coxiella burnetii antibody testing was performed and epidemiologic data were analysed from 909 servicemembers. The overall number who seroconverted to Q fever was 88 (10%). The most common ICD-9 code assigned to Q fever cases was fever not otherwise specified (NOS) (45%). A combat occupational specialty was a risk factor for Q fever seroconversion (OR = 1.8, 95% CI: 1.1–2.8) as well as receiving a primary diagnosis of fever NOS (OR = 2.6, 95% CI: 1.6–4.1).These findings indicate that Q fever is a significant infectious disease threat to military personnel deployed to Iraq. A heightened awareness among physicians is necessary to ensure prompt diagnosis and treatment.

Q fever is a zoonotic illness which frequently has a non-specific clinical presentation. Cases among deployed US military personnel have been reported in increasing numbers indicating an emerging at-risk occupational group. Banked serum specimens were utilized to estimate seroprevalence and risk factors among military personnel deployed to Iraq. Coxiella burnetii antibody testing was performed and epidemiologic data were analysed from 909 servicemembers. The overall number who seroconverted to Q fever was 88 (10%). The most common ICD-9 code assigned to Q fever cases was fever not otherwise specified (NOS) (45%). A combat occupational specialty was a risk factor for Q fever seroconversion (OR = 1.8, 95% CI: 1.1-2.8) as well as receiving a primary diagnosis of fever NOS (OR = 2.6, 95% CI: 1.6-4.1). These findings indicate that Q fever is a significant infectious disease threat to military personnel deployed to Iraq. A heightened awareness among physicians is necessary to ensure prompt diagnosis and treatment.

Callus derived from stem segments of loblolly pine (Pinus taeda L.) was subcultured to media containing 0.5% of various mono-, di-, tri- or polysaccharides. None of the carbohydrate sources tested were superior to sucrose. Growth on twelve of the 20 carbohydrates tested was more than 75% of that obtained for sucrose; growth on two other carbohydrates became >75% of the value for sucrose by the second interval on the new carbon source. Ribose and galactose-containing sugars are among those which support pine callus growth. Dramatic differences in ribose utilization are noted on media supplemented with glutamine vs. NH4/NO3 nitrogen sources.

This paper deals with problems of somatic embryogenesis (totipotency) of carrot cells in liquid culture and when dispersed in thin films of nutrient agar. The critical events that intervene between somatic cells and embryo plantlets during morphogenesis are delineated. Factors that affect the course of these events are described with special reference to the stimuli which permit the very smallest units to develop into organized structures. Special attention is directed to the importance of a protracted period of darkness and the use of an ‘embryo-conditioned medium’ as interacting stimuli that facilitate the early stages of development. Means of arresting and releasing the development of pro-embryonic units are discussed. Aspects of growth and form of the developing cultures are presented photographically at different levels. The behaviour of free protoplasts from pro-embryonic units is described and contrasted with that of intact totipotent cells. The significance of all these observations is examined in the light of practical applications and in relation to problems of development.

Five cereals and two related grasses were tested for adventitious shoot production from tissue cultures using methods concordant with those reported to be successful for cereals. The five cereals I wheat (Triticum aestivum L.), oats (Avena sativa L.), and maize (Zea mays L.) Pioneer hybrid 3369A, the Bolivian race Pororo and the Equadorian race Chococenol were all found to proliferate in culture through an aberrant root-like mechanism of growth which had the external appearance of callus. Two related species, teosinte (Zea mexicana Reeves and Mangelsdorf) and tripsacum (Tripsacum dactyloides L.), were less successful in culture, but grew in the same way. Oats, and probably Pororo and Chococeno, initiated presumptive shoot meristems directly from root vascular tissues within this root-like growth. Hybrid maize and wheat initiated no shoot meristems and produced only roots. The occasional shoot production observed in wheat was discounted as simple carryover of existing shoot apices from the primary embryo cultures. This study suggests that the incidence of shoot regeneration in cultures of these cereals may be related more directly to adventitious bud formation on roots than to any controlled de novo organogenesis from undifferentiated callus.

Tissue cultures of Zea mays L. derived from four different somatic sources all appeared to grow as callus. Microscopic examination, however, showed this growth to be merely an aberrant root-like form. A continium in the degree of aberrant root behavior, ranging from cultures appearing very root-like to those visibly indistinguishable from callus, was recognized. The continuum and ultimately the callus appearance resulted from increasing inhibition of root elongation coupled with strong enhancement of branch root initiation. This aberrant form of root growth was apparent in all cultures observed. Tissue source and growth regulator treatment interacted to shift the growth form within the extremes of the continuum but in no case was the root-like mechanism of proliferation disrupted, even during extended subculture. Removal of exogenous growth regulators from the culture medium simply allowed outgrowth of existing root primordia as normal roots and this accounts for the characteristic ease of rhizogenesis from what has been called maize callus.

THE mature vesicle of Valonia ventricosa was an early source of information about the nature of cellulose1 and the configuration of a cellulose cell wall2,3. Later it was used in one of the first investigations with the electron microscope of the way a plant cell wall develops at a naked protoplasmic surface4. The opportunity to make such a study was provided by the formation, at will, of aplanospores and by their subsequent development5. Aplanospores are small spherical masses of protoplasm, each with a nucleus, and they may form in very large numbers from the multinucleate, non-septate, protoplasm within a single vesicle. This system, therefore, has also provided an opportunity to allow sporelings to develop their cell walls while in contact with 14C-proline in sea-water. Current methods of autoradiography and electron microscopy make possible determination of the distribution of the radioactivity fixed from 14C-proline. This should test whether a developing cellulose cell wall incorporates substantial amounts of “cell wall protein” as indicated by radioactivity bound in the form of 14C-proline. Also the cell wall of mature vesicles of V. ventricosa can easily be obtained free of cytoplasm so that it is possible to ascertain, by direct analysis, how much total protein each contains and its content of proline and/or hydroxyproline.

Southern pine beetle, Dendroctonus frontalis Zimmerman, was reared aseptically from egg to adult using in vitro cultured masses (callus) of pine cells taken from loblolly pine. Callus on 3% nutrient agar was the best of the systems tried for producing aseptic beetles in 35–55 days in the complete absence of mycangial fungi. Adult production increased from 14–26% of hatched larvae when β-sitosterol was added to the culture medium.

This volume is primarily devoted to tissue culture as related to forestry. Application of tissue culture to forest species has only a short history, thus little information is available on its use for in vitro forest pathology. However, the potentials of in vitro cultures for the study of phytopathology have been demonstrated with horticultural and agricultural plants which have a rich tissue culture tradition. It is worth our time to become aware of these potentials and technologies because the in vitro approach may find its best expression in forestry where the study of pathology is hampered by tree size, rugged terrain and variable environment over the long forest life.

Conifer tissue culture had its beginnings in the late 1930s (10) and shoot regeneration cultures were first noted in 1950 (4). Since that time many species (7, 17), especially those using embryonic materials for starting expiants, have been cultured. Among some of the most studied species, Pinus radiata (2), Pseudotsuga menziesii (6), Pinus pinaster (8), Picea abies (22), and Pinus taeda (18), much laboratory data are accumulating. To date, little field data have been reported on the performance of tissue-cultured conifers (14), but several conifers are now established in field plantings (21) and data should be forthcoming.

Loblolly pine (Pinus taeda) is a good model for conifer tissue culture, since studies include three methods of in vitro propagation, long-term field evaluations of tissue-culture plantlets, and the development of in vitro trait selection methods. Abbreviated protocols are outlined for the following: (a) organogenesis of adventitious shoots and roots; (b) shoot micropropagation via fascicular and axillary shoots obtained from juvenile, adolescent, and mature explants; and (c) embryogenesis via immature zygotic embryos. Regulatory features of these processes, especially adventitious organ induction and shoot and root elongation, are examined. Field data on four- to six-year height growth, morphological characteristics, and fusiform rust resistance of tissue-culture plantlets derived from cotyledon explants are summarized from multiple plantings. Histological and immunological studies on fusiform rust resistance, evaluated in vitro in loblolly pine embryos, are presented as examples of in vitro trait selection.

Ploidy levels were calculated for callus cultures of loblolly pine (Pinus taeda L.), based on nuclear DNA content measured by Feulgen cytophotometry. The nuclear DNA content of initial stem explants showed a predominant 2C condition with less 3C and 4C, in ratios approximating those expected from diploid cells as they replicate DNA in the mitotic cell cycle. Cells with higher ploidy were produced during callus initiation, as indicated by a sharp reduction in the 2C population and a concomitant increase in higher DNA levels up to 8C. A gradual decrease in the higher ploidy levels occurred in subsequent subculture intervals, so that by 18 weeks the diploid nuclear DNA distribution was again observed, with complete elimination of DNA levels greater than 4C. Established callus cultures derived from stem or embryo explants and cultured on three different nutrient media for 48–76 weeks also showed the diploid nuclear DNA distribution with no indication of polyploid cells.

The numbers of embryogenic (S) grains present in in-situ mature anthers of Nicotiana tabacum L. were compared to the numbers of embryos and plantlets produced in cultured anthers excised at the optimal mitotic stage of development for anther culture. The Feulgen technique of staining embryos caused a considerable loss of grains from cultured anthers but this did not seriously affect the determination of the percentage of embryos present. In no instance did the numbers of embryos produced exceed the maximum number of S grains found, and the distributions of S grain and embryo frequencies in anthers were similar. In rare instances S grains which had undergone the first embryogenic division were observed in situ. The results indicate that all grains capable of embryogenesis are determined during early flower formation and that their number is not increased by in vitro culture.

Publisher Summary This chapter relates to the means by which living cells from certain vascular plants can be obtained and propagated for experimental use. Many nonvascular plants are unicellular (as are bacteria), and their culture mainly requires a proper nutrition. Hence, much physiological use has been made of some fungi and algae (e.g., Saccharomyces, Chlamydomonas, Euglena, and Chlorella). Very large cells, especially suitable for certain investigations, can be obtained from some algae (Nitella, Chara Valonia) merely by severing them from the plant or its substratum, but these are strictly multinucleate coenocytic vesicles, and, when used, they are relatively mature, sluggish in their metabolism, and do not multiply. In some cases, however, a true “free cell culture” of aplanospores can be produced easily by causing the protoplasm of the vesicle to segregate around individual nuclei. Such cultures that really start as free protoplasts, have in fact been used to study the growth of the cell wall and for 14 C-labeling experiments and in studies of ion transport and membrane potentials, but their full use in membrane-related studies has not yet been exploited.

Earlier papers of this series relate to different growth-promoting substances and systems which, singly and in combination, have interacted with trace elements (Mn and Mo) and Fe to induce growth and to affect the metabolism of aseptic cultures of carrot. The solutes of cultured carrot cells (K(+), Na(+), Cl(-), total solutes) are also affected. Two clones were grown in 9 combinations of growth factors and under 4 trace-element regimes (a complete complement including Fe, and this complement lacking either Mn or Mo, or both Mn and Mo), a total of 36 treatments under otherwise standardized experimental conditions. Under the treatments applied the number of cells varied over a 35fold range and their average size over a 7fold range; the concomitant effects on their solutes are expressed in terms of concentrations and of total content per cell. Both growth and the solutes accumulated were variously affected by carrot growth-promoting system I (mediated by inositol), by system II (mediated by IAA), and by coconut milk in the presence of Fe, with and without Mn, Mo, or Mn and Mo.The greatest concentrations of total solutes occurred in tissue cultured in nutrient solutions which lacked the stimuli to rapid cell multiplication and were also limited by the trace elements Mn and Mo. Moreover, specific regulatory effects of the trace elements on solute content, not solely attributable to their effects on cell growth, have been noted. An imbalanced growth-factor regime (zeatin acting alone, i.e. without IAA) shifted the normal preference for K(+) over Na(+) strongly toward Na(+), a trend which could also be induced by certain trace elements and more balanced growth-factor regimes, e.g. in a basal coconut milk medium lacking only Mn.The data are interpreted in the context of views on the de-novo uptake of salts and solutes in cultured cells as they grow. These cells respond to a network, or matrix, of interacting factors by distinctive effects that are attributable to the component parts of the culture medium acting singly and in various combinations. These interactions (involving trace elements and exogenous growth factors) control growth (fresh weight, number and size of cells) and regulate the solutes (organic and inorganic; K(+) vs. Na(+); organic anions vs. Cl(-)) which the cells acquire as they grow and develop. The intensity of the response of the cultures to balanced, or imbalanced, growth factors creates the internal spaces accessible to solutes; and the metabolism, as it is also affected by growth factors and trace elements, determines how these spaces are to be filled at a given osmotic value. The evidence shows the range of factors that affect the accumulation of solutes in cells as they grow and is to be contrasted with conventional observations on mature cells held in steady states under conditions that preclude all growth and when only a single ionic species is followed over a very short interval of time.