Toward Doubled Haploid Production in the Fabaceae: Progress,Constraints, and Opportunities

The Fabaceae species have a major role to play in sustainable farming systems, but they have lagged behind other families in respect to the development of doubled haploid protocols for plant improvement. Currently, no plant improvement program uses doubled haploids on a routine basis for any member of the Fabaceae. There has recently been renewed interest in haploid research as the usefulness of doubled haploid material in molecular mapping has become clear. This review provides a comprehensive account of the current information regarding the development of haploid protocols in the Fabaceae. In the Fabaceae crop species there have been isolated reports of haploid plant induction in the phaseoloid clade; soybean, cowpea and pigeonpea, as well as promising progress towards haploidy in peanut and winged bean. As yet there have been no reports of haploid plant production in the galegoid clade, but early stage haploid embryogenesis has been achieved in chickpea, field pea, and lupin. Success in the production of haploid plants has also been reported within the pasture genera Lotus, Medicago, and Trifolium and the arboreal genera Cassia, Peltophorum, and Albizzia. A review of the literature has enabled us to identify some general similarities between the protocols developed for haploid plant induction across the various legumes. These are the culture of intact anthers; use of a cold pretreatment to induce sporophytic development; targeting of microspores at the uninucleate stage of development; and use of MS (Murashige and Skoog, 1962 Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473–497. [CSA][Crossref], [Web of Science ®] [Google Scholar]) based nutrient medium with plant growth regulators to encourage continued division following induction. These protocol commonalities will assist researchers to identify approaches suited to their target Fabaceae species. The paucity of research funding for haploid research in most Fabaceae species has highlighted the need for strong collaborative linkages between institutions and researchers.

     

Isolation of plant DNA: A fast,inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.     

Micro 3D cell culture systems for cellular behavior studies: Culture matrices,devices, substrates,and in‐situ sensing methods

Microfabricated systems equipped with 3D cell culture devices and in‐situ cellular biosensing tools can be a powerful bionanotechnology platform to investigate a variety of biomedical applications. Various construction substrates such as plastics, glass, and paper are used for microstructures. When selecting a construction substrate, a key consideration is a porous microenvironment that allows for spheroid growth and mimics the extracellular matrix (ECM) of cell aggregates. Various bio‐functionalized hydrogels are ideal candidates that mimic the natural ECM for 3D cell culture. When selecting an optimal and appropriate microfabrication method, both the intended use of the system and the characteristics and restrictions of the target cells should be carefully considered. For highly sensitive and near‐cell surface detection of excreted cellular compounds, SERS‐based microsystems capable of dual modal imaging have the potential to be powerful tools; however, the development of optical reporters and nanoprobes remains a key challenge. We expect that the microsystems capable of both 3D cell culture and cellular response monitoring would serve as excellent tools to provide fundamental cellular behavior information for various biomedical applications such as metastasis, wound healing, high throughput screening, tissue engineering, regenerative medicine, and drug discovery and development.     

西双版纳乡村河溪利用方式及变化研究

西双版纳地处中国西南横断山脉向南延伸的帚状山地 ,没有气势宏大的高山峡谷 ,却有由坝子 (或沟谷 )与低山山地相间排列构成的地貌格局[1] 。贯穿在总面积 1 92 2 3× 10 4 ｋｍ2 的坝子与山地之间的是 2 76 2条大小河流 ,河网密度达 0 .6 33ｋｍ·ｋｍ- 2 。除澜沧江、罗梭江等干流和主要支流外 ,这些河流多是一些均宽不到 2 0ｍ的小河和小溪[6] 。因常年流水构成的特殊生境 ,河溪是野生动植物比较集中的地段 ,也是当地居民生活生产活动最密集的场所。河溪利用是多层次多方面的 ,如航运、水能发电 ,而使用面最广的还在乡村。西双版纳的傣族…     

Development of a novel,high-throughput screening tool for efficient perfusion-based cell culture process development

Perfusion technology has been successfully used for the commercial production of biotherapeutics, in particular unstable recombinant proteins, for more than a decade. However, there has been a general lack of high-throughput cell culture tools specifically for perfusion-based cell culture processes. Here, we have developed a high-throughput cell retention operation for use with the ambr® 15 bioreactor system. Experiments were run in both 24 and 48 reactor configurations for comparing perfusion mimic models, media development, and clone screening. Employing offline centrifugation for cell retention and a variable volume model developed with MATLAB computational software, the established screening model has demonstrated cell culture performance, productivity, and product quality were comparable to bench scale bioreactors. The automated, single use, high-throughput perfusion mimic is a powerful tool that enables us to have rapid and efficient process development of perfusion-based cell culture processes.     

Binding of flunitrazepam to differentiating neurons cultured in a chemically defined,hormone-supplemented medium

[³H]Flunitrazepam (FNZ) binding to cortical neurons from fetal rat brain was investigated in vitro. The use of a synthetic medium specific for neurons made it possible to plot a developmental curve of³H-FNZ binding in an almost pure neuronal culture. Detectable specific binding was present in vitro at time 0 (that is, the 16th gestational day). A progressive increase of binding, due to an increment in the number of recognition sites, was observed on the subsequent days. The affinity of the specific binding sites to³H-FNZ was enhanced by the addition of exogenous GABA, whereas the density was not affected.     

Isolated hepatocytes - past, present and future

The first technique for large-scale preparation of isolated hepatocytes was described in 1953 and involved perfusion of rat liver under pressure with a Ca2+-free solution containing a chelating agent. Various modifications of this technique were in use over the next ten years, until it was demonstrated that cells prepared in this manner were grossly damaged, losing most of their cytoplasmic enzymes during the preparative procedure. The successful preparation of intact isolated hepatocytes by collagenase-treatment of liver was achieved in 1967, and the widespread use of intact hepatocyte suspensions was accelerated by the development soon after of high-yield preparative techniques involving perfusion of the liver with a medium containing collagenase.The introduction of the isolated hepatocyte preparation has enabled experimental studies that otherwise would not be feasible. Important advances have been the use of cultured hepatocytes, frequently of human origin, for the investigation of the metabolism and toxicology of potential therapeutic agents. Success in this field has been achieved through the steady improvement in techniques for the maintenance in culture of differentiated hepatocytes, and in particular their cytochrome P450 complexes. Another area showing considerable promise is the employment of hepatocytes, generally from a porcine source, in temporary support systems for patients with acute liver failure. Our own studies have concentrated on the demonstration of long-range interactions between hepatocyte compartments which suggest that energy transfer between cell compartments can take place without ATP turnover.     

An improved,simple, hydroponic method for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis thaliana is one os the most studied plant model systems. Completing the genomic sequence ofA. thaliana has provided new opportunities for physiological and biochemical studies. While its small size is advantageous for genetic studies, the plant's low biomass makes it difficult to obtain enough plant material for biochemical and physiological research. The small size and rosette leaf structure, combined with the sensitivity of the apical meristem to flooding, make hydroponic growth of this model plant difficult. A few systems for hydroponic culture ofArabidopsis have been described. Gibeaut et al. (1997) introduced the use of rockwool forArabidopsis hydroponic culture. We have improved this system by introducing small-volume plastic containers with improved plugs to support the rockwool. This method is simpler than the original setup and provides improved germination and growth. The smaller containers enable the use of this system in growth chambers or small growth rooms for a large number of parallel experiments.     

It's Not the Culture of Poverty, It's the Poverty of Culture: The Problem with Teacher Education

The preparation of novice teachers is dominated by psychological notions almost to the exclusion of other social science paradigms. The perspective that is least likely to be evident in teacher preparation is that of anthropology. However, prospective and novice teachers regularly and loosely use the word "culture" as an explanation for student patterns of behavior they cannot explain. This discussion focuses on the ways prospective and novice teachers construct culture simultaneously as both the problem and the answer to their struggles with students different from themseles.     

Evaluating the toxicity of bDtBPP on CHO‐K1 cells for testing of single‐use bioprocessing systems considering media selection,cell culture volume,mixing, and exposure duration

Single‐use bioprocessing bags are gaining popularity due to ease of use, lower risk of contamination, and ease of process scale‐up. Bis(2,4‐di‐tert‐butylphenyl)phosphate (bDtBPP), a degradant of tris(2,4‐di‐tert‐butylphenyl)phosphite, marketed as Irgafos 168®, which is an antioxidant stabilizer added to resins, has been identified as a potentially toxic leachate which may impact the performance of single‐use, multilayer bioprocessing bags. In this study, the toxicity of bDtBPP was tested on CHO‐K1 cells grown as adherent or suspended cells. The EC50 (effective concentration to cause 50% cell death) for adherent cells was found to be one order of magnitude higher than that for suspended CHO‐K1 cells. While CHO‐K1 cells had good cell viability when exposed to moderate concentrations of bDtBPP, the degradant was shown to impact the viable cell density (VCD) at much lower concentrations. Hence, in developing an industry‐standard assay for testing the cytotoxicity of leachates, suspended cells (as commonly used in the bioprocessing industry) would likely be most sensitive, particularly when reporting EC50 values based on VCD. The effects of mixing, cell culture volume, and exposure duration were also evaluated for suspended CHO‐K1 cells. It was found that the sensitivity of cell culture to leachates from single‐use plastic bags was enhanced for suspended cells cultured for longer exposure times and when the cells were subjected to continuous agitation, both of which are important considerations in the production of biopharmaceuticals. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1318–1323, 2016     

Doubled haploid production in Flax (Linum usitatissimum L.)

There is a requirement of haploid and double haploid material and homozygous lines for cell culture studies and breeding in flax. Anther culture is currently the most successful method producing doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this review we focus on tissue and plants regeneration using anther culture, and cultivation of ovaries containing unfertilized ovules. The effect of genotype, physiological status of donor plants, donor material pre-treatment and cultivation conditions for flax anthers and ovaries is discussed here. The process of plant regeneration from anther and ovary derived calli is also in the focus of this review. Attention is paid to the ploidy level of regenerated tissue and to the use of molecular markers for determining of gametic origin of flax plants derived from anther and ovary cultures. Finally, some future prospects on the use of doubled haploids in flax biotechnology are outlined here.     

A simple device for planting replicate mammalian cell cultures

Summary The design and use of a unit for planting uniform inocula for replicate cultures are described. Its design permits continuous gassing of suspensions of mammalian cells with humidified CO₂, thus stabilizing the pH (±<0.05 pH unit) of culture media buffered with sodium bicarbonate. The unit can be readily modified to deliver different volumes; identical samples can be dispensed simply and rapidly, with minimal cell damage and chance of microbial contamination. Quantitative data regarding sample uniformity and growth subsequent to planting with this unit are presented.     

A simple device for planting replicate mammalian cell cultures

Summary The design and use of a unit for planting uniform inocula for replicate cultures are described. Its design permits continuous gassing of suspensions of mammalian cells with humidified CO₂, thus stabilizing the pH (±<0.05 pH unit) of culture media buffered with sodium bicarbonate. The unit can be readily modified to deliver different volumes; identical samples can be dispensed simply and rapidly, with minimal cell damage and chance of microbial contamination. Quantitative data regarding sample uniformity and growth subsequent to planting with this unit are presented.     

In vitro propagation of cauliflower,Brassica oleracea var. botrytis for hybrid seed production

Methods for obtaining heterotic F₁ and maintaining purebred lines for breeding of Brassica oleracea are limited by absence of male sterile lines and occurrence of inbreeding depression, respectively. The use of vegetative (stem, petiole, leaf, leaf rib) and floral (peduncle, pedicel, flower bud, curd) explants of cauliflower to regenerate purebred lines for crossing were examined. Of four growth regulator treatments and explant types used, best results were obtained with curd explants on MS medium with 6-benzyladenine (cytokinin) and gibberellic acid. Although 6-benzyladenine alone promoted formation of shoots in floral explants, both 6-benzyladenine and α-napthaleneacetic acid were required for vegetative explants. Use of α-napthaleneacetic acid, however, often increased callus formation. These culture techniques to maintain purebred regenerated plants will complement newly-derived nuclear-based male sterile lines obtained by the introduction of antisense copies of the gene BcpI, which is required for pollen fertility. This revised version was published online in June 2006 with corrections to the Cover Date.     

Surinamese Maroons as reggae artistes: music,marginality and urban space

This article examines how marginalized Maroon youth in Paramaribo, the capital of the Caribbean nation of Suriname, employ musical strategies in combating ethno-racial stigmatization and improving their socio-economic position. Traditionally, Maroons, after escaping the plantations during slavery, have lived in semi-isolation in Suriname's dense rainforest. In recent decades, they have become increasingly urbanized, to the discontent of many in Paramaribo, who view Maroons as backward, violent criminals. Drawing on ethnographic fieldwork and popular culture analysis, the article discusses how young Maroons use reggae and dancehall to create and recreate physical and social spaces of their own within the city and outside the forest. They protest local conditions and inequity by drawing on regional images of marginality that have been shaped by Rastafari musicians in Jamaica. Simultaneously, they use this Caribbean frame to imagine Black Atlantic unity. Connecting to global soundscapes, young Maroons strategically use music to combat their urban marginality.     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

Oncogramme,a new individualized tumor response testing method: application to colon cancer

Colon cancer is the second leading cause of cancer-related death in industrialized countries. Many anti-cancer researches are consequently performed and individualized tumor response testing (ITRT) methods are now used to individualize patient chemotherapeutic administrations. Then, a new ITRT method, Oncogramme, was developed for colon cancer. Colon tumor fragments from different patients were dissociated and seeded in a defined culture medium. Cell preparation process as well as culture medium allowed high cell viability and a good primary culture success rate. After treatment of isolated tumoral cells by chemotherapeutics alone or in combination, cytotoxicity was determined by cell death assay allowing the Oncogramme establishment, which was validated by statistical analysis. Indeed, significant results were obtained such as different profile for each patient’s cells with various drugs, and variability between patient’s cells in the response to each drug. Procedure described here to obtain the Oncogramme is a new, fast and technically reliable ITRT method applied to colon cancer. For an individualized cancer treatment use, this test should be further validated by a phase I clinical trial.     

无花果在阿图什维吾尔族民间的传统应用的调查研究

无花果(Ficus carica)是世界上驯化最早的果树之一,也是我国引入最早且现在仍在大规模种植的果树之一。新疆是我国无花果的最早种植地,也是目前种植面积最大的规模化生产地,而阿图什地区又是新疆的无花果主产区。为明确现有无花果品种,保护和传承少数民族的无花果应用传统知识,本研究采用民族植物学和植物分类学方法,对阿图什地区的无花果进行实地采集,走访调查当地维吾尔族群众,记录无花果的当地民间应用方式。结果显示阿图什地区栽培的无花果地方品种有:早熟无花果、黄无花果、小圆黄无花果及晚熟无花果等5个品种。阿图什地区维吾尔族民间流传着无花果的很多传统"民间处方",与其他植物配伍,在医药上有很多作用。此外,在庭园绿化上也大量应用无花果,具有很好的观赏效果。对阿图什地区维吾尔族民间无花果传统利用进行调研,有利于深度开发无花果资源,也将对维吾尔族植物传统知识的研究产生影响。     

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion,fibronectin-mediated adhesion,spreading, and functional activities

Summary The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture. Part of this work was presented at the 30th Annual Meeting of the Tissue Culture Association, Seattle, June, 1979.     

Influence of nutrient salts,auxins and cytokinins on the in vitro growth of Salvia greggii

Experiments were designed to determine the optimal MS salt concentration and the best auxin and cytokinin to use for shoot growth of Salvia greggii A. Gray. Full or 1/2 MS salts were superior to 1/4 MS salts based on number of shoots produced. There were no differences in the various auxins tested (IAA, NAA or IBA) as to their abilities to stimulate shoot production or increased fresh weight. BA, and BA + Kin stimulated the greatest shoot number among the cytokinins tested. A final experiment was designed to determine optimal BA and NAA concentrations for shoot growth. A medium containing 11.1M BA and no NAA produced the best growth of Salvia greggii in vitro. Shoots produced in vitro rooted and acclimatized readily in the green-house.Abbreviations MS Murashige and Skoog salts - IAA indoleacetic acid - IBA indolebutyric acid - NAA napthaleneacetic acid - BA benzyladenine - Kin kinetin - 2iP isopentenyl adenine