Gum katira – a cheap gelling agent for plant tissue culture media

Gum katira, an insoluble gum derived from the bark of Cochlospermum religiosum, has been successfully used as a gelling agent in tissue culture media for in vitro shoot formation and rooting in Syzygium cuminii and somatic embryogenesis in Albizzia lebbeck. The epicotyl segments, excised from in vitro grown seedlings of S. cuminii, developed shoots when cultured on MS medium (Murashige and Skoog, 1962), supplemented with 4% sucrose and 1 mg l^–1 BA. The so-developed shoots were rooted on Knop's medium, augmented with 2% sucrose and 1 mg l^–1 IAA. For somatic embryogenesis, hypocotyl segments derived from in vitro developed seedlings of A. lebbeck were cultured on B5 medium containing 2% sucrose. Media were gelled with either 3% gum or 0.9% agar. The quantitative response obtained on media fortified with either of the gelling agents was not significantly different. The media gelled with gum katira were almost as transparent as the liquid medium. However, viscosity of gum katira gelled medium was less than one-sixth of the viscosity of agar-gelled media, and therefore, shaking ofthe culture vessel often resulted in submersion of the explants. Nevertheless, even these submerged explants responded positively. To increase the firmness of the gum katira-gelled medium, various combinations of agar (0.2–0.6%) and gum (1–3%) were used. However, the viscosities of the media gelled with 3% gum katira as well as different concentrations of agar (0.2–0.6%) were lower than that of the medium containing only gum katira (3%). Moreover, the explant productivity obtained in neither of these combinations was more than those recorded on the control media, which were gelled either with 0.9% agar or 3% gum alone.     

Evaluation of agronomic traits in tissue culture – derived progeny of bird's-foot trefoil

Two newly selected Lotus corniculatus cultivars (Bokor and Zora), introduced recently as pasture legumes, were studied with respect to their in vitro regeneration capacity and field performance of R₁ regenerates. Multiple buds were induced in callus tissues derived from apical buds, petioles, leaf and cotyledon explants. The rooted regenerates, acclimated in a greenhouse, were fertile. Seeds from the R₁ generation were sown in the field and the plants found to be superior to controls in stem height and green mass production.     

Approaches to valuing plant medicines: the economics of culture or the culture of economics?

This paper explores the scope and limitations of the economic valuation of biodiversity. How and why is this a useful exercise, but in what way is its usefulness constrained? These issues are discussed with particular reference to cultural values within the context of medicinal plants and phytopharmaceutical development. It is argued that whilst economic valuation is an extremely important and necessary aid to formulating conservation policy, it probably is not that informative about the motivations of people in their use of certain natural resources. This is especially true for the case of medicinal plants where belief systems about the causes of disease and therefore strategies for healing are important. The paper reviews studies which have attempted to put monetary values on medicinal plants and the option values of pharmaceuticals developed from plants, and discusses the applicability of the various approaches. The implications for conservation policy, and for likely collaboration between social and natural scientists is discussed.     

Tissue culture of the desert plant Anastatica hiërochuntica

Summary Callus derived from the winter annual desert plant Anastatica hiërochuntica was grown on different media, Murashige and Skoog (1962) medium giving the best results. Large amounts of lignified xylem elements were formed resulting in an extremely hard tissue. The growth responses to different auxins, cytokinins and abscisic acid were investigated. When salts (high Na⁺, Ca²⁺ and Cl^--contents) as they can be found in aqueous extracts of desert soils from a natural A. hiëerochuntica habitat were added to Abou-Mandour (1977) or MS-media, growth of callus was inhibited drastically. In the presence of abscisic acid, however, original growth was completely restored. In salt free control media on the other hand, ABA proved to be inhibitory. Drought stress caused a decrease of both cytokinins and indoleacetic acid in the callus while ABA levels were increased, but by far not as distinct as in intact plants. Proline level was not affected by stress.Abbreviations ABA abscisic acid - AM Abou-Mandour-medium - BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DHZR dihydroxyzeatinriboside - DW dry weight - ELISA enzyme linked immuno sorbent assay - FW freshweight - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IPA isopentenyladenosine - Kin kinetin - MS Murashige and Skoog-medium     

Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean

Two relatively rare fatty acids, γ-linolenic acid (GLA) and stearidonic acid (STA), have attracted much interest due to their nutraceutical and pharmaceutical potential. STA, in particular, has been considered a valuable alternative source for omega-3 fatty acids due to its enhanced conversion efficiency in animals to eicosapentaenoic acid when compared with the more widely consumed omega-3 fatty acid, α-linolenic acid (ALA), present in most vegetable oils. Exploiting the wealth of information currently available on in planta oil biosynthesis and coupling this information with the tool of genetic engineering it is now feasible to deliberately perturb fatty acid pools to generate unique oils in commodity crops. In an attempt to maximize the STA content of soybean oil, a borage Δ⁶ desaturase and an Arabidopsis Δ¹⁵ desaturase were pyramided by either sexual crossing of transgenic events, re-transformation of a Δ⁶ desaturase event with the Δ¹⁵ desaturase or co-transformation of both desaturases. Expression of both desaturases in this study was under the control of the seed-specific soybean β-conglycinin promoter. Soybean events that carried only the Δ¹⁵ desaturase possessed a significant elevation of ALA content, while events with both desaturases displayed a relative STA abundance greater than 29%, creating a soybean with omega-3 fatty acids representing over 60% of the fatty acid profile. Analyses of the membrane lipids in a subset of the transgenic events suggest that soybean seeds compensate for enhanced production of polyunsaturated fatty acids by increasing the relative content of palmitic acid in phosphatidylcholine and other phospholipids.     

Special symposium: In vitro plant recalcitrance do free radicals have a role in plant tissue culture recalcitrance?

Summary Free radicals have an important role in the metabolism and development of aerobic organisms; however, their uncontrolled production leads to oxidative stress. This paper explores the possibility that free radical mediated stress has a role in tissue culture recalcitrance. In the context of this paper, recalcitrance is considered to be the inabilit of plant tissue cultures to respond to culture manipulations; in its broadest terms, this study also concerns the time-related decline (i.e. in vitro aging) and loss of morphogenetic competence and totipotent capacity. Studies on a diverse range of in vitro plant systems have shown that tissue cultures produce free radicals, lipid peroxides and toxic, aldehydic lipid peroxidation products. Levels of these compounds vary in response to different tissue culture manipulations, but their production is enhanced during dedifferentation and antioxidant profiles also vary throughout different phases of culture. A hypothesis is presented which suggests that tissue culture manipulations cause major metabolic and developmental changes, some of which may predispose in vitro cultures to increased free radical formation. If antioxidant protection is compromised, oxidative stress ensues and free radicals and their reaction products react with macromolecules such as DNA, proteins and enzymes, causing cellular dysfuction and as a result, the cultures become recalcitrant.     

Organ-specific expression of β-glucuronidase activity driven by the Arabidopsis heat-shock promoter in heat-stressed transgenic Nicotiana plumbaginifolia

 The expression of the Arabidopsis heat-shock protein (HSP) 18.2 promoter β-d-glucuronidase (GUS) chimera gene was studied in various organs of the transgenic Nicotiana plumbaginifolia during the recovery phase at normal temperatures (20–22  °C) following heat-shock (HS) treatment. The optimum HS temperature for GUS activity in the anthers, petals and capsules was 42  °C, but in immature seeds and the placentas of capsules it was 36  °C and 39  °C, respectively. No apparent GUS activity was observed in any organs except for dry seeds after HS at 45  °C, although the activity in dry seeds was apparent even after HS at 48  °C. After HS at 42  °C, GUS activity in the flower tissues was the highest before anthesis and declined thereafter. Received: 13 January 1998 / Revision received: 25 January 1999 / Accepted: 3 March 1999     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

CelloSelect – A synthetic cellobiose metabolic pathway for selection of stable transgenic CHO cell lines

Current protocols for generating stable transgenic cell lines mostly rely on antibiotic selection or the use of specialized cell lines lacking an essential part of their metabolic machinery, but these approaches require working with either toxic chemicals or knockout cell lines, which can reduce productivity. Since most mammalian cells cannot utilize cellobiose, a disaccharide consisting of two β-1,4-linked glucose molecules, we designed an antibiotic-free selection system, CelloSelect, which consists of a selection cassette encoding Neurospora crassa cellodextrin transporter CDT1 and β-glucosidase GH1-1. When cultivated in glucose-free culture medium containing cellobiose, CelloSelect-transfected cells proliferate by metabolizing cellobiose as a primary energy source, and are protected from glucose starvation. We show that the combination of CelloSelect with a PiggyBac transposase-based integration strategy provides a platform for the swift and efficient generation of stable transgenic cell lines. Growth rate analysis of metabolically engineered cells in cellobiose medium confirmed the expansion of cells stably expressing high levels of a cargo fluorescent marker protein. We further validated this strategy by applying the CelloSelect system for stable integration of sequences encoding two biopharmaceutical proteins, erythropoietin and the monoclonal antibody rituximab, and confirmed that the proteins are efficiently produced in either cellobiose- or glucose-containing medium in suspension-adapted CHO cells cultured in chemically defined media. We believe coupling heterologous metabolic pathways additively to the endogenous metabolism of mammalian cells has the potential to complement or to replace current cell-line selection systems.     

The use of charcoal in in vitro culture – A review

Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.     

Improvement of β-amylase thermostability in transgenic barley seeds and transgene stability in progeny

The genetic improvement of enzymes important in the brewing process is one of the main goals of barley biotechnology. For the improvement of -amylase thermostability in barley seeds, we have already constructed a mutant thermostable -amylase gene, using site-directed mutagenesis and random mutagenesis to achieve the substitution of seven amino acids of the original barley -amylase. This sevenfold-mutant barley -amylase showed a thermostability increased by 11.6 °C compared to the original enzyme. In the present article, a thermostable -amylase gene under the control of the barley -amylase promoter was introduced to barley protoplasts, and fertile plants were generated from 9 independent transgenic lines. Subsequent analyses indicated that the thermostable -amylase gene was expressed and -amylase activity derived from both native and modified genes was detected in the seeds of 6 transgenic lines. The transgene was stably transmitted to progeny, and thermostable -amylase was synthesized in T₄ seeds, demonstrating that our strategy is applicable for the improvement of seed quality for industrial utilization.     

Metabolic profiling of transgenic rice progeny using gas chromatography–mass spectrometry: the effects of gene insertion, tissue culture and breeding

The Bacillus thuringiensis ??-endotoxin and cowpea trypsin inhibitor genes have been introduced into the rice genome to improve its pest resistance via Agrobacterium-mediated transformation. A gas chromatography-mass spectrometry (GC?CMS) based metabolic profiling method was employed to determine the unpredictable metabolic changes resulting from the gene insertion and tissue culture separately. Descendants of the same transformant were obtained from different breeding programs, including both the transgenic and null-segregant progeny. The comparison of the transgenic and respective null-segregant plants enabled the evaluation of variations caused by transgenes; also the null-segregant plants were compared with the wild-type control to identify the influence of tissue culture. Based on the GC?CMS metabolic profiles, the principal component analysis and significant differences determined by Student??s t-test suggested that there were more metabolic changes from the tissue culture than those from the insertion of the transgenes. By comparing different breeding programs, it was clear that the progeny which was developed after several generations of backcross with the non-transformed rice as the recurrent parent, displayed fewer metabolic differences from the non-transformed parent. A GC?CMS based metabolic profiling study confirmed that backcrossing can help to reduce unwanted variations that occur during transformation processes.     

Effects of the cucumber mosaic virus 2a protein on aphid–plant interactions in Arabidopsis thaliana

The cucumber mosaic virus (CMV) 2a RNA-dependent RNA polymerase protein has an additional function in Arabidopsis thaliana, which is to stimulate feeding deterrence (antixenosis) against aphids. Antixenosis is thought to increase the probability that aphids, after acquiring CMV particles from brief probes of an infected plant's epidermal cells, will be discouraged from settling and instead will spread inoculum to neighbouring plants. The amino acid sequences of 2a proteins encoded by a CMV strain that induces antixenosis in A. thaliana (Fny-CMV) and one that does not (LS-CMV) were compared to identify residues that might determine the triggering of antixenosis. These data were used to design reassortant viruses comprising Fny-CMV RNAs 1 and 3, and recombinant CMV RNA 2 molecules encoding chimeric 2a proteins containing sequences derived from LS-CMV and Fny-CMV. Antixenosis induction was detected by measuring the mean relative growth rate and fecundity of aphids (Myzus persicae) confined on infected and on mock-inoculated plants. An amino acid sequence determining antixenosis induction by CMV was found to reside between 2a protein residues 200 and 300. Subsequent mutant analysis delineated this to residue 237. We conjecture that the Fny-CMV 2a protein valine-237 plays some role in 2a protein-induced antixenosis.     

Arabidopsis thaliana as a model for the study of plant–virus co-evolution

Understanding plant–virus coevolution requires wild systems in which there is no human manipulation of either host or virus. To develop such a system, we analysed virus infection in six wild populations of Arabidopsis thaliana in Central Spain. The incidence of five virus species with different life-styles was monitored during four years, and this was analysed in relation to the demography of the host populations. Total virus incidence reached 70 per cent, which suggests a role of virus infection in the population structure and dynamics of the host, under the assumption of a host fitness cost caused by the infection. Maximum incidence occurred at early growth stages, and co-infection with different viruses was frequent, two factors often resulting in increased virulence. Experimental infections under controlled conditions with two isolates of the most prevalent viruses, cauliflower mosaic virus and cucumber mosaic virus, showed that there is genetic variation for virus accumulation, although this depended on the interaction between host and virus genotypes. Comparison of Q_ST-based genetic differentiations between both host populations with F_ST genetic differentiation based on putatively neutral markers suggests different selection dynamics for resistance against different virus species or genotypes. Together, these results are compatible with a hypothesis of plant–virus coevolution.     

Fungal culture of musculoskeletal tissue: what’s the point?

There have not been any studies that review the prevalence of fungal isolates using selective media from samples of banked musculoskeletal tissue retrieved from living and cadaveric donors. A total of 2,036 swab and 2,621 biopsy samples of musculoskeletal tissue from tissue banks were received from the 1st August 2008 till 31st December 2010. Routine culture for fungi using selective media with a prolonged incubation period failed to demonstrate a greater prevalence of fungal isolates than by using non-selective culture media alone. Using selective culture fungi were recovered from only two Sabouraud agar plates (0.1%) but not from non-selective media. During the same period fungi were isolated from three graft samples cultured in non-selective broth media only (0.1%). There was no correlation of fungal isolates from selective or non-selective media inoculated at the same time nor from multiple graft samples collected from the same donor supporting the possibility of an exogenous source for fungal isolates rather than an endogenous source.     

Biochemical properties and mechanism of action of a vanadyl(IV) – aspirin complex on bone cell lines in culture

A recently synthesized vanadyl(IV) complex with aspirin[VO(aspirin)ClH₂O]₂, has been thoroughly investigated by physicochemical techniques. In order to support the proposed structure, stoichiometry and the coordination sphere of the vanadium center, some studies such as elemental analysis, electronic (diffuse reflectance) and vibrational (infrared) spectroscopies, magnetic susceptibility, as well as the thermal behavior, were carried out. The bioactivity of the vanadium complex (VOAspi) was evaluated on two osteoblast-like cell lines in culture, being its cytotoxic effects stronger than the vanadyl cation as assessed by morphological changes and lipid peroxidation. These effects may be partially explained through the induction of the expression of Erks (Extracellular signal-regulated kinases) and the inhibition of the PTPases (Phosphotyrosine phosphatases) present in the cellular extracts.