Micropropagation ofAcacia mearnsii

Summary A micropropagation system was developed forAcacia mearnsii De Wild., which is the principal source of the world’s tanbark and an excellent firewood. Shoot tips 5-mm long from 3-wk-old seedlings germinated in vitro served as explants. The seeds were germinated on hormone-free MS medium and the shoot tips were cultured on three-fourth-strength MS medium supplemented with combinations of auxins [indole-3-butyric acid (IBA) andα-naphthaleneacetic acid (NAA)] and cytokinins [kinetin and benzylaminopurine (BAP)]. Cultures were maintained at 25° ± 5° C and exposed to 12-h photoperiods of cool-white fluorescent light (70 μEm⁻²·s⁻¹). Multiple shoot formation was promoted by BAP at 2 mg · liter⁻¹ (8.87μM) and higher combined with or without 0.01 mg · liter⁻¹ (0.049μM) IBA. Cytokinins at concentrations of less than 1 mg · liter⁻¹ combined with 0.01 to 0.1 mg · liter⁻¹ auxin inhibited multiple shoot formation and promoted rooting of the shoot tip explants. Shoot multiplication cultures were maintained by transferring segments of multiple-shoot clusters onto a medium containing 2 mg · liter⁻¹ BAP and 0.01 mg · liter⁻¹ IBA. Although higher levels of BAP promoted more multiple shoot formation, this BAP level allowed shoot elongation as well as multiplication. In-vitro-produced shoots were induced to root on a range of NAA concentrations (0.0 to 0.8 mg · liter⁻¹[4.3μM]) supplemented to half- or full-strength MS medium. The highest frequency of root proliferation was on half-strength MS medium supplemented with 0.6 mg · liter⁻¹ (3.22μM) NAA. Plantlets survived in potting soil and exhibited normal growth under greenhouse conditions.     

Bioengineering Considerations for a Nurturing Way to Enhance Scalable Expansion of Human Pluripotent Stem Cells

Understanding how defects in mechanotransduction affect cell‐to‐cell variability will add to the fundamental knowledge of human pluripotent stem cell (hPSC) culture, and may suggest new approaches for achieving a robust, reproducible, and scalable process that result in consistent product quality and yields. Here, the current state of the understanding of the fundamental mechanisms that govern the growth kinetics of hPSCs between static and dynamic cultures is reviewed, the factors causing fluctuations are identified, and culture strategies that might eliminate or minimize the occurrence of cell‐to‐cell variability arising from these fluctuations are discussed. The existing challenges in the development of hPSC expansion methods for enabling the transition from process development to large‐scale production are addressed, a mandatory step for industrial and clinical applications of hPSCs.     

Culture of human corpus cavernosum endothelium

Summary A method for culturing endothelial cells (HCC-EC) from surgical specimens of human corpus cavernosum has been developed. The approach involves selective endothelial outgrowth from explants and may be generally applicable to tissue whose endothelium is not amenable to isolation by routine mechanical or enzymatic methods. The tissue is minced into pieces which are placed onto gelatin-or fibronectin-coated tissue culture plastic, and grown in medium suitable for microvascular endothelial cell growth (Carson and Haudenschild, In Vitro 22:344–354, 1986). By Days 5 to 7 EC colonies are found. Within a day or two after the appearance of the EC colonies, a non-EC cell type appears and, if undisturbed, quickly overgrows the EC. An exploitable temporal separation between the emergence of EC and non-EC is obtained when both conditioned medium (from bovine aortic endothelium) and retinal extract are present during the outgrowth period. Explants are removed by pipetting at the first sign of the emergence of the non-EC cell type. Once isolated, HCC-EC do not require conditioned medium but do require either retinal extract or acidic fibroblast growth factor for survival and growth. Approximately 60% of the first passage cultures are at least 80% EC as judged by DiI-Ac-LDL labeling. One corpus (0.3×0.3×0.5 cm) usually produces 120 cm² of primary culture within 2 wk. These EC form contact-inhibited monolayers and stain positively for Factor VIII. They have a doubling time at 6th passage of 48 h and a plateau density of 5 to 7×10⁴ cells/cm². The availability of such cultures should facilitate the study of endothelium-mediated responses which play an important role in the erectile function of human penile corpus cavernosum. Supported by NIH R01 HL23567-09, DK-39080-01, DK40025-01, DK40487-01.     

A simple cell disrupter designed for small cells with relatively large nuclei

A simple cell disrupter that is particularly suitable for breaking small cells with relatively large nuclei is described. Cells are disrupted by the shearing forces set up as they are pushed by a positive N2 pressure through a 0.8- to 1.5-micrometer stainless-steel filter. The total procedure takes only a few minutes, it is highly reproducible, and the yield is good. The cell homogenate obtained is a good starting source for the isolation of plasma membranes, intracellular organelles, and nuclei.     

Changing arabinosylation patterns of wall-bound hydroxyproline in bean cell cultures

The arabinosylation patterns of wall-bound hydroxyproline in Phaseolus vulgaris L. cell suspension cultures were determined by separating free hydroxyproline and hydroxyproline-arabinose oligomers over a Bio-Gel P-2 column. Total hydroxyproline accounted for about 3.3% of wall dry weight during all growth phases of batch-cultured bean cells. The chemical arabinosylation patterns of wall-bound hydroxyproline varied during the lag phase and early log phase of the culture. First, an increase in nonglycosylated hydroxyproline occurred accompanied by a corresponding decrease in hydroxyproline tetra-arabinoside. During the early log phase the reverse happened. In later stages of growth the chemical arabinosylation patterns remained constant. The radiochemical arabinosylation patterns were also determined, after pulselabeling the cultures with [¹⁴C]proline at various times during growth, to be able to distinguish recently incorporated hydroxyproline. The time course of the arabinosylation pattern of this fraction indicated that the initial changes in the chemical pattern were due to the temporary incorporation of less extensively glycosylated hydroxyproline-containing protein into the cell wall.Abbreviations Hyp hydroxyproline - HA_n hydroxyproline arabinoside - with n arabinosyl residues - TFA trifluoroacetic acid     

Sugarcane mosaic virus in plantlets regenerated from diseased leaf tissue

Plantlets produced from sugarcane leaf tissue were examined to determine the effect of propagation on the frequency of occurrence of sugarcane mosaic virus (SCMV).Explants from immature leaf tissues of the sugarcane variety CP 72-356 (Saccharum interspecific hybrid), healthy or SCMV-infected, were cultured on Murashige-Skoog medium to which a combination of cytokinin and auxin had been added. Plantlets developed on healthy and infected leaf tissue within 6 weeks. The juice from plantlets was assayed for SCMV on Rio sorghum (Sorghum bicolor (L.) Moench, var. Rio) seedlings and on sugarcane varieties CP 31-294 and CO 31-588 for SCMV-strain identification. Results indicated that SCMV strain H was transmitted from the donor tissue to the regenerated plantlets. Observation on plantlets reared in the greenhouse showed that 23% had symptoms of SCMV. In a second replicated experiment, the leaf tissue from plants of POJ 234 free of mosaic or infected with SCMV strain A, B, D, H, or I was cultured. Each of the five strains was transmitted from donor to plantlet as indicated by assays on sorghum and sugarcane varieties. From 11 to 88% of the plantlets had mosaic symptoms, depending on the strain infecting the donor plant. In this experiment, SCMV-strain M was transmitted from an unidentified donor variety to 23% of the regenerated plantlets.Portions of this paper have been presented to the American Society of Sugar Cane Technologists, at the meeting in Clearwater, Florida in June, 1984.     

Genetic instability during embryogenic cloning of celery

Genetically marked tissues of celery (Apium graveolens) were employed to contrast genetic and chromosomal stability in serially bulk-transferred callus and regenerated plants. After six months in culture, 84% of the callus cells were karologically indistinguishable from normal, while the remainder exhibited chromosome loss and/or fusion. All of 50 clones derived from this tissue expressed the control phenotype with respect to heterozygous isozyme markers. Of 95 plants regenerated from the same tissue, 94 were phenotypically indistinguishable from the original explant donor, and cytogenetic analyses revealed the presence in 4.3% of an accessory chromosome, while the remainder were normal diploids. Analysis of the selfed progeny of these regenerated plants revealed the presence of a new recessive mutation causing abnormal leaf morphology at a frequency of 1.8%. Only one of 40 cells in 12-month-old callus tissue was karyologically indistinguishable from normal, the remainder consisting primarily of hypodiploids. The observation that all 50 clones were phenotypically heterozygous was statistically inconsistent with the hypothesis that hypodiploidy was associated with random complete chromosome loss. The culture had, at this point, lost the ability to regenerate. It is speculated that embryogenic cloning of celery may be suitable under certain circumstances for direct field establishment, but that levels of new genetic variation are sufficiently high to preclude its use for seed production.     

Separation of precultured pollen from Nicotiana tabacum by aqueous polymer two-phase partition

Pollen isolated from cold treated and precultivated anthers of tobacco (Nicotiana tabacum L. var. Wisconsin 38) were separated into different fractions with counter-current distribution using an aqueous Dextran-polyethylene glycol two-phase system. It was possible to distinguish among eight pollen classes differing in developmental stage and in partitioning. A part of each fraction was cultivated for analysis of embryo formation. This was highest in a fraction with an intermediate to high partition in the phase system. Enriched in this fraction were also pollen that were fairly well stained with acetocarmine, contained several nuclei and had a relatively low mitochondrial activity. The enrichment of embryogenic pollen offers several advantages especially to physiological studies on embryogenesis.     

Methane production from propionate by methanogenic mixed culture

Abstract The association of Desulfobulbus sp. with Methanosarcina barkeri 227 was able to produce CH₄ from propionate in the presence of sulfate, if a sufficient amount of ferrous iron was added to the media in order to trap the soluble sulfides produced from sulfate. In the absence of ferrous iron, soluble sulfides inhibited the acetoclastic reaction. Attempts to cultivate Desulfobulbus sp. with H₂-utilising methanogenic bacteria in the absence of sulfate did not succeed.