A novel technique to overcome browning in tissue culture

Experiments conducted using Dioscorea alata L. revealed that an exudate from the cut end of the explants was responsible for browning of the culture medium. Browning did not affect growth of roots and shoots when explants were cultured in a large volume of medium, but in a small volume it was lethal. Sealing the cut ends with paraffin wax was found to control browning by preventing exudation. This simple technique permitted establishment of cultures in a small volume of medium in about 90 percent of the cases, while in unsealed cultures lethal browning was recorded in 80 percent of the cases. The advantages of this technique over other methods of controlling browning are discussed.     

Diffusion of phosphate to plant roots in soil

Summary A method is described for computing the theoretical distribution of solutes around a root with root hairs. The P concentration profile around the primary root of a rape seedling with root hairs, growing in a low-P soil, showed considerably greater depletions than predicted by this method, using the relationship between P in soil and in solution obtained from a desorption isotherm experiment. This suggests an enhanced release of soil P into solution in the rhizosphere.Soil Science Laboratory, Department of Agricultural Science, University of Oxford     

The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts

Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites was still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Three-dimensional electron microscopy data showed that sporozoites enter salivary gland cells through a ring-like structure and by forming a transient vacuole. The absence of a functional AMA1-RON complex led to an altered morphology of the entry junction, associated with epithelial cell damage. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to efficiently and safely enter the mosquito salivary glands to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.     

Construction of a Genetic Linkage Map and Identification of QTLs for Seed Weight and Seed Size Traits in Lentil (Lens culinaris Medik.)

Seed weight and seed size both are quantitative traits and have been considered as important components of grain yield, thus identification of quantitative trait loci (QTL) for seed traits in lentil (Lens culinaris) would be beneficial for the improvement of grain yield. Hence the main objective of this study was to identify QTLs for seed traits using an intraspecific mapping population derived from a cross between L. culinaris cv. Precoz (seed weight-5.1g, seed size-5.7mm) and L. culinaris cv. L830 (seed weight-2.2g, seed size-4mm) comprising 126 F₈-RILs. For this, two microsatellite genomic libraries enriched for (GA/CT) and (GAA/CTT) motif were constructed which resulted in the development of 501 new genomic SSR markers. Six hundred forty seven SSR markers (including 146 previously published) were screened for parental polymorphism and 219 (33.8%) were found to be polymorphic among the parents. Of these 216 were mapped on seven linkage groups at LOD4.0 spanning 1183.7cM with an average marker density of 5.48cM. Phenotypic data from the RILs was used to identify QTLs for the seed weight and seed size traits by single marker analysis (SMA) followed by composite interval mapping (CIM) which resulted in one QTL each for the 2 traits (qSW and qSS) that were co-localized on LG4 and explained 48.4% and 27.5% of phenotypic variance respectively. The current study would serve as a strong foundation for further validation and fine mapping for utilization in lentil breeding programs.     

Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties

Several decades back ascorbic acid was proposed as an effective anticancer agent. However, this idea remained controversial and the mechanism of action unclear. In this paper, we show that ascorbic acid at a concentration reported to be achievable through high doses of oral consumption is capable of cytotoxic action against normal cells. Several antioxidants of both animal as well as plant origin including ascorbic acid also possess prooxidant properties. Copper is an essential component of chromatin and can take part in redox reactions. Previously we have proposed a mechanism for the cytotoxic action of plant antioxidants against cancer cells that involves mobilization of endogenous copper ions and the consequent generation of reactive oxygen species. Using human peripheral lymphocytes and Comet assay we show here that ascorbic acid is able to cause oxidatative DNA breakage in normal cells at a concentration of 100–200 μM. Neocuproine, a Cu(I) specific sequestering agent inhibited DNA breakage in a dose dependent manner indicating that Cu(I) is an intermediate in the DNA cleavage reaction. The results are in support of our above hypothesis that involves events that lead to a prooxidant action by antioxidants. The results would support the idea that even a plasma concentration of around 200 μM would be sufficient to cause pharmacological tumor cell death particularly when copper levels are elevated. This would account for the observation of several decades back by Pauling and co-workers where oral doses of ascorbic acid in gram quantities were found to be effective in treating some cancers.     

Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek)

Limited availability of validated, polymorphic microsatellite markers in mung bean (Vigna radiata), an important food legume of India, has been a major hurdle towards its improvement and higher yield. The present study was undertaken in order to develop a new set of microsatellite markers and utilize them for the analysis of genetic diversity within mung bean accessions from India. A GA/CT enriched library was constructed from V. radiata which resulted in 1,250 putative recombinant clones of which 850 were sequenced. SSR motifs were identified and their flanking sequences were utilized to design 328 SSR primer pairs. Of these, 48 SSR markers were employed for assessing genetic diversity among 76 mung bean accessions from various geographical locations in India. Two hundred and thirty four alleles with an average of 4.85 alleles per locus were detected at 48 loci. The polymorphic information content (PIC) per locus varied from 0.1 to 0.88 (average: 0.49 per locus). The observed and expected heterozygosities ranged from 0.40 to 0.95 and 0.40 to 0.81 respectively. Based on Jaccard’s similarity matrix, a dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) analysis which revealed that one accession from Bundi, Rajasthan was clustered out separately while remaining accessions were grouped into two major clusters. The markers generated in this study will help in expanding the repertoire of the available SSR markers thereby facilitating analysis of genetic diversity, molecular mapping and ultimately broadening the scope for genetic improvement of this legume.     

Endogenously expressed estrogen receptors mediate neuroprotection in hippocampal cells (HT22)

Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism.