Showing papers by "Sumita Jha published in 2017"

Biotechnological Approaches for Production of Anti-Cancerous Compounds Resveratrol, Podophyllotoxin and Zerumbone.

Abstract: Secondary metabolites from numerous plant sources have been developed as anti- cancer reagents and compounds such as resveratrol, podophyllotoxin and zerumbone are of particular importance in this regard. Since their de novo chemical synthesis is both arduous and commercially expensive, there has been an impetus to develop viable, biotechnological methods of production. Accordingly, this review focuses on the recent developments in the field, highlighting the use of micropropagation, cell suspension cultures, callus cultures, hairy root cultures, recombinant microbes and genetically modified higher plants. Optimization of media and culture conditions, precursor feeding, immobilization and the use of chemical or physical elicitation in various protocols has led to an increase in resveratrol and podophyllotoxin production. Heterologous gene transformation of higher plants with stilbene synthase derived from Arachis hypogaea or Vitis vinifera lead to resveratrol production with the concomitant increase in resistance to plant pathogens. Interestingly, genetic transformation of Podophyllum hexandrum and Linum flavum with Agrobacterium rhizogenes resulted in Ri-T-DNA gene(s)-mediated enhancement of podophyllotoxin production. Zerumbone yields from tissue cultured plantlets or from suspension cultures are generally low and these methods require further optimization. In microbes lacking the native resveratrol or zerumbone biosynthesis pathway, metabolic engineering required not only the introduction of several genes of the pathway, but also precursor feeding and optimization of gene expression to increase their production. Data pertaining to safety and toxicity testing are needed prior to use of these sources of anti-cancer compounds in therapy.

Effects of cryptogein gene on growth, phenotype and secondary metabolite accumulation in co-transformed roots and plants of Tylophora indica

Abstract: Tylophora indica, an indigenous medicinal plant, was transformed with the cryptogein gene to determine the effect of crypt gene on secondary metabolites in co-transformed roots and plants via Agrobacterium rhizogenes mediated transformation. The Ri crypt co-transformed roots and plants showed expression of crypt gene. Southern hybridization specifies that crypt gene has been transferred and positively integrated into the Ri crypt co-transformed plant. AFLP fingerprinting revealed high degree of genetic similarity among the Ri-transformed and Ri crypt co-transformed cultures. The expression of crypt gene stimulated phenolic compound accumulation in transformed root and plants while tylophorine content was comparable in Ri transformed and Ri crypt co-transformed root lines and plants. The Ri crypt co-transformed root lines showed significantly higher (p ≤ 0.05) phenolics production (caffeic acid, 1.8–2.9-fold; p-coumaric acid, 1.9-fold and ferulic acid, 1.5–2-fold) compared to Ri-transformed root lines. The roots of Ri crypt co-transformed plants showed a significantly (p ≤ 0.05) higher content of caffeic acid (1.19-fold) and ferulic acid (1.53-fold) than Ri-transformed plants. It is suggested that crypt-transformed plants can also be used as a tool to elucidate the biochemical basis of defense responses as phenolics are known to play a role in providing defense barriers to infection by pathogen.

Morpho-histological characterization and direct shoot organogenesis in two types of explants from Bacopa monnieri on unsupplemented basal medium

Abstract: In the present study, high frequency regeneration has been obtained via de novo direct shoot organogenesis from leaf and internode explants in Murashige and Skoog (MS) basal medium without any phytohormone supplementation in Bacopa monnieri, an indigenous traditionally used medicinal herb. Leaves and internodes from different positions were excised from 4-weeks-old in vitro propagated B. monnieri plants and cultured on MS basal medium supplemented with 3% (w/v) sucrose and 0.75% (w/v) agar for 4 weeks. The induction of de novo shoot buds was observed at petiolar cut edges of leaf and both proximal and distal cut ends of internode explants within 10–15 days of culture. The first histological changes could be observed after 4–5 days, with meristematic activity of vascular bundles. Proliferation of epidermal cells gave rise to dome-shaped protuberances followed by shoot apical meristems formation and their vascular connections with explant tissues within 2 weeks of culture. However, a basipetal gradient of shoot regeneration from both types of explants collected along the branch axis was noticed after 4 weeks of culture. Leaf and internode explants near the basal region exhibited significantly higher number of shoot buds and micro shoots (8.8/leaf explant and 15/internode explant). Microshoots (7–12 micro shoots/leaf or internode explants) elongated (shoot length 8–9 cm) within 8 weeks on phytohormone free MS medium. Excised micro shoots rooted (100%) in hormone free MS medium within two weeks of culture. Rooted plants were then acclimatized and transferred to field with 95% survival. This protocol may be used for micropropagation, genetic transformation as well as a model system for evaluation of changes associated with acquisition of competence of differentiated cells in phytohormone free medium.

Transgenesis and Secondary Metabolism

Abstract: The first € price and the £ and $ price are net prices, subject to local VAT. Prices indicated with * include VAT for books; the €(D) includes 7% for Germany, the €(A) includes 10% for Austria. Prices indicated with ** include VAT for electronic products; 19% for Germany, 20% for Austria. All prices exclusive of carriage charges. Prices and other details are subject to change without notice. All errors and omissions excepted. S. Jha (Ed.) Transgenesis and Secondary Metabolism

Chromosome morphometric analysis of Indian cultivars of Lens culinaris Medik. using EMA based Giemsa staining method

Abstract: The influence of biotic, abiotic and climatic changes on crop production is becoming increasingly evident. Gaps in demand and supply of lentils, one of the few protein-rich crops in India, are gradually increasing. Of seven estimated species of lentil, six are wild and the other, Lens culinaris, is the only species under cultivation, with a large number of cultivars. Chromosome analysis is beneficial to breeders and genome researchers in crop improvement programmes. However, a chromosomal database of this important crop is not available in India. The present paper has described a detailed chromosome analysis of 21 certified Indian cultivars of L. culinaris using enzymatic maceration and air drying (EMA) based Giemsa staining methods for the first time. Uniform chromosomal preparations have resulted in variation in average chromosome size (4.94–9.8 μm), total chromatin length (69.18–137.24 μm), and satellite bearing chromosome number (third, fourth and fifth) among the cultivars. Though our results...

Ribosomal DNA ITS1, 5.8S and ITS2 secondary structure, nuclear DNA content and phytochemical analyses reveal distinctive characteristics of four subclades of Protasparagus

Abstract: The use of ribosomal DNA (rDNA) internal transcribed spacer (ITS) primary sequence based phylogeny is a conventional practice to estimate the evolutionary interspecies relationship. However, analysis of the functional folding patterns and higher order secondary structures of ITS regions can provide additional important information regarding species relatedness and interspecies variations. In the present study, we provide the first detailed information on the rDNA ITS secondary structure diversity in the four subclades of the subgenus Protasparagus. Several angiospermic conserved motifs were identified in each of the ITS1, 5.8S and ITS2 secondary structures of the studied taxon. Topological comparison of the ITS1 secondary structures showed variations in the helix- IV regions. Moreover, presence of unique sequence motifs and differences in the internal loop structures were found to be subclade specific. The present study suggests that comprehensive analysis of the ITS1, 5.8S and ITS2 structural elements including helices, loops and bulges can be used as an important tool for species delimitation. The present study investigated the evolution of the secondary structure of ITS marker (its phylogenetic utility), genome size, base chromosome number and phytochemicals, and identified a putative polyploid event shared by a number of Protasparagus species. The phytochemical analysis of two important active compounds, i.e., shatavarin-IV and sarsasapogenin, also reveals their presence in all the studied taxa constitutively even at the subclade level.

Flow Cytometry and Its Utility

Abstract: Flow cytometry is a laser-based biophysical technique to study optical properties of microscopic particles in fluid suspension. The technology is not only routinely used in medicine (transplantation, hematology, cancer, prenatal diagnosis, genetics, and sperm sorting for sex preselection) but also has many other applications in basic research, crop improvement, clinical practice, and clinical trials. The principles of flow cytometric chromosome analysis and sorting known as flow cytogenetics and research in this field have increased over the past four decades due to high sensitivity and precision of this technique. The use of nucleic acid-specific fluorochromes has semiautomated quantitative chromosome analysis, thus reducing subjectivity of preexisting slide-based methods. Flow cytometric classification of chromosomes (flow karyotyping) enables detection of chromosomal aberrations, while flow sorting permits isolation of single chromosome types in large quantities to be used for gene mapping, preparation of chromosome-specific gene libraries, and ultimately sequencing leading to chromosome genomics. Flow fluorescent in situ hybridization (FISH), i.e., combined fluorescent in situ hybridization in suspension (FISHIS) and flow cytometry using microsatellite DNA or gene-specific probes, is another approach to increase our knowledge on structure, function, and evolution of chromosomes. These techniques provide increased value for diagnosis and management of clinical diseases, especially cancer which is generally characterized by high chromosomal instability. Flow cytogenetics also has important applications in plant biology, especially in the study of genome evolution and crop improvement. This review outlines the utility of flow cytometry in chromosomal analysis, its applications, limitations, and future directions.

A proteomic approach to evaluate the effects of endogenous expression of cryptogein gene in crypt-transgenic plants of Bacopa monnieri

Abstract: Cryptogein, a proteinaceous elicitor has been reported to affect growth and secondary metabolism in several plant species. We established two transgenic plant lines of Bacopa monnieri L., an indigenous medicinal species, following Agrobacterium tumefaciens mediated transformation. The transgenic plant lines obtained following transformation with strains LBA4404-nptII-crypt (plant line BmAt-ncrypt) and LBA4404-nptII (plant line BmAt-n), were maintained on selection media i.e. MS medium containing 100mg/l Kanamycin. To evaluate the effects of endogenous expression of cryptogein gene in crypt-transgenic plants of B. monnieri, in the present study, a proteomic approach was employed. Using 2D-PAGE followed by MALDI TOF MS/MS, we found 54 spots with reproducible differences in abundance (≥+2 or ≤-0.5-fold) between the plant lines. Bioinformatics analyses helped their identification, functional annotation and categorization. Significantly upregulated gene expression (p≤0.5) of most of the key enzymes of triterpenoid saponin biosynthetic pathway (mevalonate kinase, mvk; mevalonate diphosphate decarboxylase, mdc; squalene synthase, sqs; β-amyrin synthase, bas and UDP-glycosyl transferase 2, ugt 2) in crypt-transgenic plant lines (BmAt-ncrypt) of B. Monnieri was observed as compared to non-transformed (Bm-NT) and empty vector control (BmAt-n) plant lines. Cryptogein-induced ROS production, a typical stress response of plant cells towards cryptogein, was significantly increased (p≤0.5) in specific activities and higher abundance of several anti-oxidant enzymes, such as SOD (3.0-5.0-fold), APX (2.3-3.0-fold) and CAT (2.6-fold) in BmAt-ncrypt. The upregulation of CaM (1.6-fold) and calcium-transporting ATPase-2 (2.0-fold) in BmAt-ncrypt indicate possible calcium-dependant cryptogein elicitation. A 3.0-fold increase in EIN3 levels with down-regulation of F-box protein, its regulator also indicate induction of JA-ET signalling pathway in crypt-transgenic plants. This study not only demonstrates involvement of multiple defence signalling pathways in crypttransgenic plants of B. monnieri but also shows first evidence of the involvement of JA-ET signalling in cryptogein-induced elicitation.

Fluorescent Chromosome Banding and Genome Size Estimation in Three Species of Swertia

Abstract: Chromosomal attributes of the critically endangered medicinal plant Swertia chirayita along with its allied species S. nervosa and S. bimaculata are precisely essential to assess phylogenetic relationships. The present study deals with karyomorphometric analysis based on patterns of fluorescence chromosome banding and nuclear genome size estimation by flow cytometry reported for the first time in S. chirayita (2n=26), S. nervosa (2n=26) and S. bimaculata (2n=26). Fluorescent banding revealed the distinct differences in chromosomal CMA+ve/DAPI-ve heterochromatic sites among the species. There were six chromosomes with CMA+ve signals in S. chirayita and two chromosomes with CMA+ve signals in S. bimaculata whereas all chromosomes of S. nervosa displayed CMA+ve signals. Due to a difference in CMA banding pattern, proportion of GC rich regions differed among the chromosomes of S. chirayita (3.32%), S. nervosa (18.76%), and S. bimaculata (0.64%). S. bimaculata had the highest 2C DNA content (9.82 pg/2C) compared to S. nervosa (1.95 pg/2C) and S. chirayita (1.09 pg/2C). Lowest amount of GC-rich CMA sites along with highest DNA content distinguished S. bimaculata from the other two species. A nearly nine-fold difference in nuclear genome size among the homoploid species of Swertia (2n=26) and particular chromosomal discrimination by localized CMA bands allowed interpretation of karyotypic affiliations in the three species of Swertia.