Profiling C6-C3 and C6-C1 phenolic metabolites in Cocos nucifera

This paper reports the detection and identification of phenolic metabolites (C6-C3 and C6-C1 compounds) in Cocos nucifera. An HPLC/UV system was used to analyze the soluble and wall-associated phenolics in mesocarp and leaf tissues of C. nucifera. Alkaline hydrolysis of the cell wall material of the mesocarpic and leaf tissues yielded 4-hydroxybenzoic acid as the major phenolic compound. Other phenolic acids identified were ferulic acid, 4-coumaric acid, 4-hydroxybenzaldehyde and vanillic acid. No significant qualitative differences in composition were observed between leaf and mesocarp, but there were quantitative variations in the metabolite levels.     

Expression and affinity purification of recombinant proteins from plants

With recent advances in plant biotechnology, transgenic plants have been targeted as an inexpensive means for the mass production of proteins for biopharmaceutical and industrial uses. However, the current plant purification techniques lack a generally applicable, economic, large-scale strategy. In this study, we demonstrate the purification of a model protein, beta-glucuronidase (GUS), by employing the protein calmodulin (CaM) as an affinity tag. In the proposed system, CaM is fused to GUS. In the presence of calcium, the calmodulin fusion protein binds specifically to a phenothiazine-modified surface of an affinity column. When calcium is removed with a complexing agent, e.g., EDTA, calmodulin undergoes a conformational change allowing the dissociation of the calmodulin-phenothiazine complex and, therefore, permitting the elution of the GUS-CaM fusion protein. The advantages of this approach are the fast, efficient, and economical isolation of the target protein under mild elution conditions, thus preserving the activity of the target protein. Two types of transformation methods were used in this study, namely, the Agrobacterium-mediated system and the viral-vector-mediated transformation system.     

An intact plant assay for estimating nitrogenase activity (C2H2 reduction) of sorghum and millet plants grown in pots

Summary A non destructive intact-plant assay for estimating nitrogenase activity (C₂H₂ reduction) of pot-grown sorghum and millet plants is described. Plants with intact shoots sustained more activity than plants whose tops were removed prior to the assay. With this technique individual plants can be assayed several times during their life cycle. The C₂H₂ reduction was linear up to 16h incubation in this assay procedure. More rapid diffusion of C₂H₂ was achieved by injection through a Suba seal in the bottom of the pot. The equlibration of injected C₂H₂ in the gas phase of the pots filled with sand and sand:FYM media was completed within 1 h. Significantly higher nitrogenase activity and better growth of sorghum and millet plants occurred when plants were grown in a mixture of sand and farmyard manure (FYM) than when plants were grown in vermiculite, soil, or sand + soil medium. Nitrogenase activity and plant growth were greater in a mixture of sand with 2 and 3% FYM than with 0.5 and 1% FYM. Activity was higher when the plants were incubated at 33°C and 40°C than at 27°C. Activity also increased with increasing soil moisture. There were significant differences amongst 15 sorghum cultivars screened for associated nitrogenase activity. This new technique has good prospects for screening cultivars of millet, sorghum and other grain crops for their nitrogen-fixing ability.Submitted as Journal article No. 358 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Charcot-Marie-Tooth disease 1A (CMT1A) associated with a maternal duplication of chromosome 17p11.2→12

We report here the second case of Charcot-Marie-Tooth disease 1A (CMT1A) with a cytogenetically visible de novo direct duplication of 17p11.117p12. A male child who was initially referred for developmental delay and dysmorphism was subsequently shown to have significantly reduced motor nerve conduction velocities characteristic of CMT1A. This patient was not informative for the DNA markers mapping to the CMT1A region; however, with DNA markers pA10–41 and EW503 that map proximally and distally with respect to the disease locus, a dosage difference was observed between the two alleles. Comparison with parental genotypes indicated a de novo maternal duplication. Pulsed field gel analysis using probe VAW409R3a indicated that a 500-kb SacII junction fragment usually associated with CMT1A was absent in this patient. These findings confirm that the disease phenotype is probably caused by a gene dosage effect.     

The mapping of chromosome 4q markers in relation to facioscapulohumeral muscular dystrophy (FSHD).

The Basque population is one of the oldest populations of Europe. It has been suggested that the Basques arose from a population established in western Europe during the late Paleolithic Age. The Basque language (Euskera) is a supposedly pre-Indo-European language that originates from the first settlers of Europe. The variable distribution of the major cystic fibrosis (CF) mutation (delta F508 deletion) in Europe, with higher frequencies of the mutation in northern Europe and lower frequencies in southern Europe, has suggested that the delta F508 mutation was spread by early farmers migrating from the Middle East during the Neolithic period. We have studied 45 CF families from the Basque Country, where the incidence of CF is approximately 1/4,500. The birthplaces of the parents and grandparents have been traced and are distributed according to their origin as Basque or Mixed Basque. The frequency of the delta F508 mutation in the chromosomes of Basque origin is 87%, compared with 58% in those of Mixed Basque origin. The analysis of haplotypes, both with markers closely linked to the CF gene and with intragenic markers, suggests that the delta F508 mutation was not spread by the Indo-European invasions but was already present in Europe more than 10,000 years ago, during the Paleolithic period.     

Growth and organogenesis in moth bean callus cultures as influenced by triazole growth regulators and gibberellic acid

The triazole plant growth regulators, paclobutrazol and uniconazole, reduced in vitro growth of moth bean callus by 20–25% when added to the culture medium at 1 mg/L (3.4 μM). The addition of 10 mg/L (29 μM) gibberellic acid (GA₃) to the culture medium in combination with the triazoles restored callus growth to a level equivalent to that of the untreated control. GA₃ alone had little effect on callus growth. When added to a regeneration medium at 1 mg/L both paclobutrazol and uniconazole reduced the percentage of cultures that formed roots, as well as the mean number of roots per culture. In contrast, GA₃ increased root formation and counteracted the inhibitory effects of the triazoles on rooting. The addition of triazoles or GA₃ to the regeneration medium reduced the formation of green meristematic nodules, which are precursors of shoots in moth bean callus. When callus was grown in the presence of either paclobutrazol or uniconazole, subsequent root and green meristematic nodule formation were reduced upon transfer to a growth regulator-free regeneration medium. The results of this study indicate that exposure of moth bean callus tissue to micromolar concentrations of triazoles or GA₃ can significantly alter in vitro growth and differentiation.     

Influence of growth regulators on somatic embryogenesis,plantlet regeneration,and post-transplant survival of Echinochloa frumentacea

After placement on Murashige and Skoog's basal medium supplemented with 3–5 mg/l 2,4-D, immature inflorescence expiants of Echinochloa frumentacea gave rise to three distinct types of callus: a) loosely arranged and soft; b) compact and translucent; c) compact, sticky and mucilaginous. Somatic embryo formation occurred in type b callus in about 18–24 d. Callus types a and c did not produce somatic embryos. The highest percentage of cultures exhibiting somatic embryogenesis occurred on the medium containing 5 mg/l 2,4-D and 0.5 mg/l kinetin. Somatic embryos also formed directly on the inflorescence (without intervening callus formation) in about 15% of the expiants placed on this medium. The addition of paclobutrazol or uniconazole (0.25 or 1 mg/l) to the medium had no influence on the percentage of cultures exhibiting direct somatic embryogenesis, but paclobutrazol slightly increased the mean number of somatic embryos per culture. Many of the callus-derived somatic embryos germinated when subcultured on basal MS medium supplemented with kinetin. Addition of paclobutrazol or uniconazole to the culture medium at 0.25 or 1 mg/l decreased somatic embryo germination and shoot elongation but increased root length and leaf width. Both paclobutrazol and uniconazole increased survival of the plantlets following transplanting to soil. Increased post-transplant survival was accompanied by reduced water loss from plantlets produced on culture media containing triazoles.