Raju Bharalee

In Vitro Clonal Propagation of Curcuma caesia Roxb and Curcuma zedoaria Rosc from Rhizome Bud Explants

Two species of Curcuma (C. caesia and C. zedoaria) have been propagated through tissue culture using rhizome bud explant. The best response for shoot multiplication was obtained on MS basal medium supplemented with 4 mg l−1 BAP and 1.5 mg l−1 NAA for C. caesia (3.5 ± 0.79 shoots per explant) and 1 mg l−1 BAP + 0.5 mg l−1 NAA for C. zedoaria (4.5 ± 0.15 shoots per explant). A maximum of 9.2 ± 0.15 and 8.9 ± 0.09 roots per explant were obtained for C. caesia and C. zedoaria, respectively when MS was supplemented with 0.5 mg l−1 IAA. The rooted plants could be established in soil.

Identification of drought tolerant progenies in tea by gene expression analysis.

Understanding the genes that govern tea plant (Camellia sinensis) architecture and response to drought stress is urgently needed to enhance breeding in tea with improved water use efficiency. Field drought is a slow mechanism and the plants go through an adaptive process in contrast to the drastic changes of rapid dehydration in case of controlled experiments. We identified a set of drought responsive genes under controlled condition using SSH, and validated the identified genes and their pattern of expression under field drought condition. The study was at three stages of water deficit stress viz., before wilting, wilting and recovery, which revealed a set of genes with higher expression at before wilting stage including dehydrin, abscissic acid ripening protein, glutathione peroxidase, cinnamoyl CoA reductase, calmodulin binding protein. The higher expression of these genes was related with increase tolerance character of DT/TS-463 before wilting, these five tolerant progenies could withstand drought stress and thus are candidates for breeding. We observed that physiological parameter like water use efficiency formed a close group with genes such as calmodulin related, DRM3, hexose transporter, hydrogen peroxide induced protein, ACC oxidase, lipase, ethylene responsive transcription factor and diaminopimelate decarboxylase, during wilting point. Our data provides valuable information for the gene components and the dynamics of gene expression in second and third leaf against drought stress in tea, which could be regarded as candidate targets potentially associated with drought tolerance. We propose that the identified five tolerant progenies on the basis of their drought tolerance can thus be utilised for future breeding programmes.

Production of biodiesel from freshwater microalgae and evaluation of fuel properties based on fatty acid methyl ester profile

ABSTRACT Eleven freshwater microalgal strains of Chlorella sorokiniana BTA4015; Chlorella sp. BTA4032, C. variabilis BTA4036; Chlorella sp. BTA4071; C. variabilis BTA4109; C. variabilis BTA4121; Chlorella sp. BTA4146; Chlamydomonas asymmetrica BTA4028; C. asymmetrica BTA4154; Scenedesmus armatus BTA4076 and Scenedesmus regularis BTA4112 were isolated from northeast India and identified through internal transcribed spacer 2 region sequence alignment. Algal strains cultured in 20 L carboys in outdoor condition in tap water enriched with NPK showed doubling time ranged between 1.5–6.8 d, lipid content (4.5–9.1% dry wt), biomass productivity (6–26 mg L−1 d−1) and lipid productivity (0.31–1.99 mg L−1 d−1). The biodiesel properties of iodine value, saponification value, cetane number, high heating value, oxidation stability, allylic and bis-allylic position equivalent, kinematic viscosity, cold filter plugging point, cloud point, pour point and C18:3 were calculated from fatty acid profile. Equally weighted parameters for biodiesel component using Preference Ranking Organization Method for Enrichment Evaluation and Graphical Analysis for Interactive Assistance analysis ranked Chlorella variabilis BTA4109 & BTA4036, C. sorokiniana BTA4015 the most suitable strains meeting the EN14214 biodiesel standard. The biomass productivity for the three strains recorded 20.83, 26.04 and 6.83 mg L−1 d−1 whereas the lipid productivity recorded 0.95, 1.89 and 0.32 mg L−1 d−1 respectively.

Molecular Landscape of Helopeltis theivora Induced Transcriptome and Defense Gene Expression in Tea

Tea is the second most consumed beverage worldwide whose cultivation is greatly challenged by a large variety of biotic and abiotic stresses. Among the biotic factors, the hemipteran pest Helopeltis theivora Waterhouse is particularly devastating in Asia and Africa, rendering the crop unsuitable for any downstream processing. The present study, for the first time, endeavors to dissect the molecular events associated with such infestations and identify potential molecular protagonists influencing Helopeltis tolerance in Assam tea. We analyzed the transcriptome of infested tea plants having contrasting responses to pest feeding by suppression subtractive hybridization and quantified the relative abundances of defense-associated transcripts. We analyzed 445 unigenes derived from 1558 expressed sequence tags (ESTs) from three different infestation conditions. Our study indicate that defense responses to the pest greatly vary in temporal and cultivar-specific manner characterized by an exclusive upregulation of specific defense-associated genes in the tolerant cultivar. Moreover, it was observed that transcripts related to flavonoid biosynthesis, purine metabolism, formate metabolism, jasmonic acid biosynthesis and signaling, and cell wall metabolism are uniquely enriched in the same. Interestingly, those involved in caffeine biosynthesis remain comparatively low and unaltered. We report higher trichome densities on the abaxial surface of the leaf in tolerant cultivar while puncture causes their tender reorientation. SEM of the insect mouthparts reveal higher concentrations of sensilla-like structures near the distal end and tip of the proboscis which are known to act as chemoreceptors in other hemipterans. Our investigation provides the first ever glimpse of Helopeltis-induced defense gene network and provides the much needed platform for improvement of the crop for better Helopeltis tolerance through breeding and transgenic approaches. On a wider perspective, the study will augment our understanding of molecular responses to phloem herbivory in plants.

Bioinformatics tools for development of fast and cost effective simple sequence repeat (SSR), and single nucleotide polymorphisms (SNP) markers from expressed sequence tags (ESTs)

The development of current molecular biology techniques has led to the generation of huge amount of gene sequence information under the expressed sequence tag (EST) sequencing projects on a large number of plant species. This has opened a new era in crop molecular breeding with identification and/or development of a new class of useful DNA markers called genic molecular markers (GMMs). These markers represent the functional component of the genome in contrast to all other random DNA markers (RMMs). Many recent studies have demonstrated that GMMs may be superior to RMMs for use in the marker assisted selection, comparative mapping and exploration of functional genetic diversity in the germplasms adapted to different environment. Therefore, identification of DNA sequences which can be used as markers remains fundamental to the development of GMMs. Amongst others; bioinformatics approaches are very useful for development of molecular markers, making their development much faster and cheaper. Already, a number of computer programs have been implemented that aim at identifying molecular markers from sequence data. A revision of current bioinformatics tools for development of genic molecular markers is, therefore, crucial in this phase. This mini-review mainly provides an overview of different bioinformatics tools available and its use in marker development with particular reference to SNP and SSR markers. Keywords: Genic molecular marker, simple sequence repeat (SSR), and single nucleotide polymorphisms (SNP) markers from expressed sequence tags (ESTs). African Journal of Biotechnology Vol. 12(30), pp. 4713-4721

Transgenic Tea Over-expressing Solanum tuberosum Endo-1,3-beta-d-glucanase Gene Conferred Resistance Against Blister Blight Disease

Tea (Camellia sinensis [L.] O. Kuntze) plant, one of the most important plantation crops in the world, is infected by a fungus called Exobasidium vexans leading to dreaded blister blight disease. The disease may result in crop losses up to 35% which directly affect the tea industry. Solanum tuberosum endo-1,3-beta-d-glucanase was cloned into tea genome via Agrobacterium-mediated transformation. The transformation event initially gave 32 kanamycin-resistant plantlets, out of which PCR analysis confirmed only 10 plantlets about the integration of transgene in the plant genome. Real-time PCR study detected transgene expression in six transgenic plantlets. Upregulation of endogenous C. sinensis pathogenesis-related (PR) genes like PR3 (chitinase I) gene and PR5 (thaumatin-like protein) gene also occurred in transgenic plantlets. Detached leaf infection assay showed resistance to E. vexans in greenhouse-acclimated transgenic plantlets. An inhibitory activity against E. vexans was noticed on the detached leaves of transgenic plantlets compared to control. Transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area unlike the formation of fungal lesion on control plantlet. Thus, it can be inferred that constitutive expression of the potato endo-1,3-beta-d-glucanase gene can be a strategy to produce blister blight-resistant tea.

Molecular cloning, expression and computational analysis of a water stress inducible copper-containing amine oxidase gene (CuAO) from tea plant [ Camellia sinensis (L.) O. Kuntze]

Copper-containing amine oxidase (CuAO) is the enzyme known to play diversity of function in plant responses to environmental stresses through its reaction products. Here, for the first time we report full length cDNA encoding CuAO protein from a drought tolerant tea cultivar. It was found to be 785 bp long with a 70 bp 5.-UTR, 193 bp 3.-UTR, 522 bp mORF and a polyA adenylational signal. It codes for a poly-peptide of 173 amino acids having predicted molecular weight and isoelectric point of 19 KDa and 7.75 respectively. Heterologous expression and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein in Escherichia coli revealed similar size as predicted by in silico analysis. Blastp analysis and template based homology modeling in Phyre2 has identified a copper amine oxidase domain with ligand binding site for copper at residue 123 (Histidine) which suggests its probable role in plant responses to environmental stresses. Interestingly, no signal peptide sequence was detected in the predicted protein which is in contrast to the CuAO so far reported in plants. Although, in slico analysis of the protein have indicated its probable structure and functions, further functional characterization is needed to better understand its role during drought and other environmental stresses in tea. Key words: Camellia sinensis , copper amine oxidase, homology modeling, molecular cloning.