An improved micropropagation protocol for teak

An improved micropropagation protocol has been developed for teak (Tectona grandis). Nodal explants placed on MS medium supplemented with 22.2 M benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (76.8%). Establishment was reduced when explants were retained in the initial culture medium longer than 12 h. Explants collected in May showed maximum (76.8%) response. Placement of the explants on MS medium supplemented with 22.2 M benzylaminopurine and 0.57 M indole-3-acetic acid resulted in the maximum average number of shoots. In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (9.8 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture. This treatment resulted in 77.9% survival of the plantlets. They were weaned in a glasshouse and finally moved to an agro-net shade house.     

Purification and differentiation of three avian adenoviruses by restriction endonuclease analysis

A rapid method of ultracentrifugation pelleting of avian adenovirus (AAV) from small volume of chloroform treated infected cell culture fluid or allantoic fluid was adapted for isolation of adenoviral DNA. The viral DNA extracted from semipurified viruses was found to be intact on agarose gel and pure enough (A260/280 = 1.85-1.92) for restriction enzyme analysis. Restriction endonuclease analysis of Indian strain of AAV serotype 1, AAV serotype 4 (group I AAVs) and egg drop syndrome-76 (EDS-76) virus genomes (group III AAV) with Hind III enzyme differentiated these viruses. The AAV serotype 1 and serotype 4 strain exhibited identical Hind III profile to European viral strains belonging to same serotypes however, the EDS-76 virus gave similar but not identical profile. The calculated genomic lengths for AAV serotype 1 and EDS-76 virus were approximately found to be 33.9 and 44.4 Kb, respectively.     

Molecular and morphological characterization of somatic hybrids between <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. and <Emphasis Type="Italic">S. etuberosum</Emphasis> Lindl.

Interspecific potato somatic hybrids between Solanum tuberosum L. (di)haploid C-13 and 1 endosperm balance number non-tuberous wild species S. etuberosum Lindl. were produced by protoplasts electrofusion. The objective was to transfer virus resistance from this wild species into the cultivated potatoes. Post-fusion products were cultured in VKM medium followed by regeneration of calli in MS₁₃ K medium at 20°C under a 16-h photoperiod, and regenerants were multiplied on MS medium. Twenty-one somatic hybrids were confirmed by RAPD, SSR and cytoplasm (chloroplast/mitochondria) type analysis possessing species-specific diagnostic bands of corresponding parents. Tetraploid nature of these somatic hybrids was determined through flow cytometry analysis. Somatic hybrids showed intermediate phenotypes (plant, leaves and floral morphology) to their parents in glass-house grown plants. All the somatic hybrids were male-fertile. ELISA assay of somatic hybrids after artificial inoculation of Potato virus Y (PVY) infection reveals high PVY resistance.     

Baculovirus expression system: An alternative for producing catalytically active human PTP-1B

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovi-rus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.     

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.     

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa)

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na⁺/K⁺ ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5–18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na⁺/K⁺ ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.