Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Proteomic approach for identification and characterization of novel immunostimulatory proteins from soluble antigens of Leishmania donovani promastigotes

Visceral leishmaniasis (VL) caused by Leishmania donovani is a major parasitic disease prevalent in endemic regions of Bihar in India. In the absence of good chemotherapeutic options, there is a need to develop an effective vaccine against VL which should be dependent on the generation of a T helper type 1 (Th1) immune response. We have shown that soluble proteins from promastigote of a new clinical isolate of L. donovani (2001) ranging from 68 to 97.4 kDa (F2 fraction), induce Th1 responses in the peripheral blood mononuclear cells of cured Leishmania patients and hamsters and also showed significant prophylactic potential. To understand the nature of F2 proteins, it was further characterized using 2-DE, MALDI-TOF and MALDI-TOF/TOF-MS. In all, 63 spots were cut from a CBB stained gel for analysis and data was retrieved for 52 spots. A total of 33 proteins were identified including six hypothetical/unknown proteins. Major immunostimulatory proteins were identified as elongation factor-2, p45, heat shock protein (HSP)70, HSP83, aldolase, enolase, triosephosphate isomerase, protein disulfideisomerase and calreticulin. This study substantiates the usefulness of proteomics in characterizing a complex protein fraction (F2) map of soluble L. donovani promastigote antigen identified as Th1 stimulatory for its potential as vaccine targets against VL.     

Antifungal activity of some marine organisms from India,against food spoilage Aspergillus strains

Crude aqueous methanol extracts obtained from 31 species of various marine organisms (including flora land faunal), were screened for their antifungal activity against food poisoning strains of Aspergillus. Seventeen species exhibited mild (+ =zone of inhibition 1–2 mm) to significant (+++ =zone of inhibition 3–5 mm) activity against one or the other strain under experiment. However, extracts of 12 species were active against all the three strains. Organisms like Salicornia brachiata(obligate halophyte), Sinularia leptocladus(Soft coral), Elysia grandifolia (Mollusks),Gorgonian sp. 2 and Haliclona sp. exhibited significant (inhibition zone of 3–5 mm) antifungal activity against one or the other strains. However,extracts of A. ilicifolius, Amphiroa sp.,Poryphyra sp., Unidentified sponge, Suberites vestigium, Sinularia compressa,Sunularia sp., Sinularia maxima, Subergorgia suberosa, Echinogorgia pseudorassopo and Sabellaria cementifera were mild (inhibition zone of 1–2 mm) to moderate(inhibition zone of 2–3 mm) active against the respective strains. The growth of A. japonicuswas significantly inhibited by the extracts ofS. leptocladus (r = 0.992, p < 0.0001)and E. grandifolia (r = 0.989, p < 0.0001).This revised version was published online in October 2005 with corrections to the Cover Date.     

Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity

The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.     

Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD

RAPD based fingerprinting of 21 serovars of Bacillus thuringiensis (Bt) representing different serotypes was performed using 19 random decamer primers. A total of 172 polymorphic fragments, ranging in size from 161-2789 bp, were amplified from 13 of the 19 primers. Pairwise genetic similarity analysis revealed very low similarity values, ranging from 3-68%, among the serovars of Bt, indicating high genetic divergence. Nineteen serovars of Bt fell in two major clusters and remaining two formed solitary clusters in the dendogram. Clustering of Bt strains established genetic relatedness between serovars and serotypes. It has been suggested that RAPD analysis can be used for genotypic characterization of Bt to complement flagellar serotyping.     

Development of doubled haploids from an elite <Emphasis Type="Italic">indica</Emphasis> rice hybrid (BS6444G) using anther culture

A total of 200 doubled haploids (DHs) were generated from an elite rice hybrid, ‘BS6444G’ for which an androgenic method was developed by manipulating the physical and chemical factors. The spike pretreated at 10?°C for 7–8 days was effective for callusing and green plant regeneration. The maximum callus frequency was achieved when the anthers cultured in N6 medium supplemented with 2.0 mg L^?1 2,4-diclorophenoxyacetic acid, 0.5 mg L^?1 6-benzylaminopurine and 3% maltose. Calli induced in N6 media also showed significant green shoot regeneration in MS medium supplemented with 0.5 mg L^?1 1-napthalene acetic acid, 0.5 mg L^?1 kinetin, 1.5 mg L^?1 benzylaminopurine and 3% sucrose producing 210 green plants. Assessment of the ploidy status showed 95.71% fertile diploids and 4.2% polyploids; no haploids were observed. A total of 38 sequence-tagged microsatellite (STMS) markers proved able to discriminate a heterozygote from all the 200 DHs. The DHs grown in the field showed significant variation for their agronomic traits. Comparison of traits with control indicates homogeneity within each DH line and significant variance of traits between DH lines. Nine DH lines produce higher grain yield than the hybrid parent which suggests the possibility of exploiting hybrid vigor in indica rice through the development of DH lines of high yielding hybrids.