Tapas Kumar Bandyopadhyay

Stevioside Induced ROS-Mediated Apoptosis Through Mitochondrial Pathway in Human Breast Cancer Cell Line MCF-7

Stevioside is a diterpene glycoside found in the leaf of Stevia rebaudiana, a traditional oriental medicinal herb, which has been shown to have various biological and ethno-medicinal activities including antitumor activity. In this study, we investigated the effects of stevioside on the cytotoxicity, induction of apoptosis, and the putative pathways of its action in human breast cancer cells (MCF-7). For the analysis of apoptotic pathway, measurement of reactive oxygen species (ROS) and assessment of mitochondrial transmembrane potential (MTP) were achieved. We showed that stevioside was a potent inducer of apoptosis and it conveyed the apoptotic signal via intracellular ROS generation; thereby inducing change in MTP and induction of mitochondrial mediated apoptotic pathway. Taken together, our data indicated that stevioside induces the ROS-mediated mitochondrial permeability transition and results in the increased expression of apoptotic proteins such as Bax, Bcl-2 and Caspase-9. Effect of stevioside on stress-related transcription factors like NF-E2-related factor-2 opens up a new vista for further studies. This is the first report on the mechanism of the antibreast cancer (in vitro) activity of stevioside.

Erratum to: Direct somatic embryogenesis and genetic homogeneity assessment of regenerated plants of Anthurium andraeanum Linden cv. Fantasia

We present here a method for the in vitro propagation of Anthurium andraeanum cv. Fantasia through direct somatic embryogenesis. The embryos were directly formed at the cut end of most of the leaf explants when cultured on MS basal medium supplemented with N6-benzyladenine (BA) plus α-naphthalene acetic acid (NAA). MS basal media supplemented with BA (0.27 μM) and NAA (2.68 μM) induced maximum number of somatic embryos per leaf explant (15.33 ± 1.45) after 28 d of continuous culture. The highest numbers of embryos (12.33 ± 0.88) were matured into plantlets in MS basal medium augmented with 0.27 μM BA and 58.4 mM sucrose. Histology showed the presence of scutellum, coleoptile, well-developed root, and shoot initials at different stages of somatic embryo (SE) development directly on leaf explants. About 85% of the plantlets were successfully acclimatized, and of these, 80.3% plants survived after secondary hardening under greenhouse conditions. The embryo-derived plants successfully flowered. The presence of monomorphic DNA bands in random amplified polymorphic DNA (RAPD) marker analysis confirmed the genetic homogeneity of direct somatic embryo-derived plantlets in respect to their mother plant.

Identification and Validation of a New Male Sex-Specific ISSR Marker in Pointed Gourd (Trichosanthes dioica Roxb.)

The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb.) to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR) primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR) amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp) was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.

Hormonal control of sex expression of cucumber (Cucumis sativus L.) with the identification of sex linked molecular marker

Sex differentiation in plants is closely related to the process of plant growth and development. The mechanism that regulates the transition in flowering plants from the vegetative state to reproductive development is poorly understood because the flowering physiology is difficult to study using traditional methods. In the present study, tissue culture system was used as a potential tool to study the sex determination of cucumber to reduce the influence of environmental factors. Major role of gibberellic acid and cytokinins in the regulation of sex expression of cucumber were demonstrated. Gibberellic acid was associated with male sex expression whereas cytokinins found to be associated with the female sex expression. However 6-Benzyl adenine was found to induce hermaphrodite flowers. In the present study, in vitro flowering with different sex expression as well as sex conversion correlates the established fact that minor changes in the balance between phytohormones in the plants with the same genotypic conditions can result in the development of one sex or another. To understand the molecular background of sex expression in cucumber, random amplified polymorphic DNA (RAPD) marker technique was adopted. The result shows unique banding pattern for genome fingerprinting at an early stage of analysis. Two sex linked markers namely OPS250-11 and OPS600-11 were scored for male and bisexual individuals respectively, that are helpful to understand the developmental biology and stable sex expression of cucumber.

Electrophoresis-staining apparatus for DNA agarose gels with solution exchange and image acquisition

ABSTRACT Agarose gel electrophoresis is routinely used for the separation of nucleic acids. Gels are developed, stained, and visualized using dedicated equipment and reagents. Manufacturers have developed instrumentation with advanced features that provide good safety and user-friendly operation. However, the process of size fractionation of nucleic acids by horizontal gel electrophoresis by dye staining may be cumbersome and unsafe due to many steps and harmful chemicals. Here is reported a safe, inexpensive, time-saving, and comprehensive apparatus for gel electrophoresis, staining, and imaging. This newly modified apparatus has simple operation and uses existing equipment and off-the-shelf components for easy construction in the laboratory. The apparatus has been shown to perform agarose horizontal gel electrophoresis and associated techniques with ease and simplicity.

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver–Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

A tetrad apparatus for protein gel casting, electrophoresis, staining, and scanning techniques with dual sensors for automatic detection of gel polymerization and protein migration

Recent advancements in biochemical sciences have facilitated researchers to explore the structure and function of macro molecules in a cell. PAGE is one of the most favored and adapted laboratory techniques. Due to its simple and economical procedures, several variants or new modifications are routinely observed in the basic electrophoresis technique that comprises gel casting, electrophoresis, staining, and imaging process which consequently necessitates additional apparatuses/components in the laboratory. Operation of these additional apparatuses/components lengthens the pre‐ and postelectrophoresis procedures involving many intermittent tedious and time‐consuming steps. A universal apparatus that can facilitate all such associated techniques is lacking and is of utmost importance for fast and effective results. An apparatus that can perform synchronized action of slab gel casting (16 × 16 cm), electrophoresis (SDS‐PAGE), dye staining (Coomassie), and imaging (scanning) techniques with real‐time monitoring through sensor technology is described in this article. The estimated cost (∼

Simple and rapid system for two‐dimensional gel electrophoresis technique: A laboratory exercise for high school students

150) of fabrication of the apparatus is very economical and simple assembly procedure of the main apparatus can be completed within ∼30 min after fabrication.

Application of molecular markers in plant genome analysis: a review

Polyacrylamide gel electrophoresis (PAGE) is the most classical technique favored worldwide for resolution of macromolecules in many biochemistry laboratories due to its incessant advanced developments and wide modifications. These ever‐growing advancements in the basic laboratory equipments lead to emergence of many expensive, complex, and tricky laboratory equipments. Practical courses of biochemistry at high school or undergraduate levels are often affected by these complications. Two dimensional gel electrophoresis technique (2D‐PAGE) used for resolving thousands of proteins in a gel is a combination of isoelectric focusing (first dimension gel electrophoresis technique) and sodium‐dodecylsulphate PAGE (second dimension gel electrophoresis technique or SDS‐PAGE). Two different laboratory equipments are needed to carry out effective 2D‐PAGE technique, which also invites extra burden to the school laboratory. Here, we describe a low cost, time saving and simple gel cassette for protein 2D‐PAGE technique that uses easily fabricated components and routine off‐the‐shelf materials. The performance of the apparatus was verified in a practical exercise by a group of high school students with positive outcomes.

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

Advancement in the field of molecular biology has led to the development of various molecular markers which has revolutionized our understanding of the organization and evolution of plant genomes. Detection of genetic variation in plants offers an opportunity to understand the molecular basis of several biological phenomena. The reliability and efficiency of restriction digestion and polymerase chain reaction based random DNA markers have already proved their utility in taxonomical, evolutionary and ecological studies of plants. Progresses in the field of genomics and transcriptomics have enabled plant researchers to develop molecular markers derived from exon region of the genome which are termed as genic molecular markers (GMMs). GMMs are the part of the cDNA/EST sequences that mainly characterize the functional part of the genome. Next-generation DNA sequencing has also significantly contributed towards development of microRNA specific novel functional markers at the DNA level. This review focuses on the technical aspects of different molecular markers and their applications in the genome analysis.