Krishnan Kathiravan

Adventitious regeneration in vitro occurs across a wide spectrum of squash (Cucurbita pepo) genotypes

Cucurbita pepo L. (squash, pumpkin) is a highly polymorphic vegetable species of major importance. Our study characterized a spectrum of C. pepo germplasm for the ability to regenerate in vitro by direct organogenesis from cotyledon explants. Cultivars tested included both cultivated subspecies, texana and pepo, and nearly all of their respective cultivar-groups. Direct shoot regeneration occurred in all accessions, and was generally high (56–94%), with a single exception of 22% (‘Bolognese’). There was no significant difference between the percentage regeneration of the two subspecies. Shoot regeneration per responding explant was uniform (1.2–1.6 shoots per explant). Only ‘True French’ produced statistically more shoots (3.9 per explant) than other accessions. The morphology of regeneration varied. Most cultivars produced long shoots, often fasciated, amid a few small buds. Some subspecies pepo cultivars (Beirut, Yugoslavia 7, Ma’yan and True French) produced short, massive, hollow shoots, sometimes accompanied by shoots that were more normal. Two subspecies texana cultivars (Creamy Straightneck and Small Bicolor) produced single (sometimes double) shoots without other buds. The production of chimeric (mixoploid) regenerants varied and there was a tendency to regenerate chimeric plants from the widest-fruited accessions (i.e. lowest length-to-width ratio) in each subspecies. Subspecies pepo Pumpkin Group ‘Tondo di Nizza’ showed significantly greater production of chimeric regenerants. In comparison with the great range of variation observed in fruit shape, the variation of in vitro responses (mostly less than 2-fold in regeneration and shoot production) was less than expected.

DNA templated self-assembly of gold nanoparticle clusters in the colorimetric detection of plant viral DNA using a gold nanoparticle conjugated bifunctional oligonucleotide probe

The DNA templated self-assembly of gold nanoparticles clustered in different configurations (n = 2–∞) was investigated in the colorimetric detection of ToLCNDV DNA using a gold nanoparticle conjugated bifunctional oligonucleotide probe. The AuNP-bifunctional oligonucleotide probe conjugate was prepared using citrate capped AuNPs (∼19 nm) and virus specific ssoligo probes 1 and 2. Each conjugate consisted of ∼105 ssoligo probes 1 and 2, specific for the forward and reverse strands of the PCR amplified ToLCNDV dsDNA target. The intensity of the UV-visible absorbance spectra of AuNP-bifunctional oligonucleotide probes decreased gradually after hybridization with increasing ratios of dsDNA targets (0.0–1.0). Hybridized AuNP-bifunctional oligonucleotide probes showed gradual resistance against salt induced aggregation with increasing concentrations of dsDNA target, up to the ratio of 1.0. The color of solution remained red even after hybridization of the AuNP-bifunctional oligonucleotide probe with the dsDNA target. These hybridized AuNP-bifunctional oligonucleotide probes were found as clusters with different configurations (n = 2–11) and defined interparticle distances (1.3–2.1 nm). This target DNA guided the self-assembly of the AuNP-bifunctional oligonucleotide probes which is the reason for the different optical absorbance properties of hybridized solutions before and after the salt treatment. An exciting finding of this investigation is that the AuNP-bifunctional oligonucleotide probes were anchored on the center core particle, tending to form a AuNP cluster that incorporated from 1–6 Au-NP probes and extended to 8 and 10 with an increasing size of core particle diameter. The assembly of three dimensional DNA templated AuNP clusters in flower and pyramid shapes was possible with a dsDNA target and a AuNP-bifunctional oligonucleotide probe but not with a AuNP-monofunctional oligonucleotide probe. The limit of detection of dsDNA target in this bifunctional nanoprobe assay was ∼7.2 ng. Also, this AuNP-bifunctional oligonucleotide probe could reduce the concentration of target DNA required for colorimetric detection by half, as it could recognise both strands in the dsDNA target simultaneously. The proof of this concept will be used for further development of ultra sensitive nanoassay methods and will also be applicable for materials science applications.

Gold nanoparticles assisted characterization of amine functionalized polystyrene multiwell plate and glass slide surfaces

We demonstrated citrate-capped gold nanoparticles assisted characterization of amine functionalized polystyrene plate and glass slide surfaces through AuNPs staining method. The effect of AuNPs concentration on the characterization of amine modified surfaces was also studied with different concentration of AuNPs (ratios 1.0–0.0). 3-Aminopropylyl triethoxy silane has been used as amine group source for the surface modification. The interactions of AuNPs on modified and unmodified surfaces were investigated using atomic force microscopy and the dispersibility, and the aggregation of AuNPs was analyzed using UV–visible spectrophotometer. Water contact angle measurement and X-ray photoelectron spectroscopy (XPS) were used to further confirmation of amine modified surfaces. The aggregation of AuNPs in modified multiwell plate leads to the color change from red to purple and they are found to be adsorped on the modified surfaces. Aggregation and adsorption of AuNPs on the modified surfaces through the electrostatic interactions and the hydrogen bonds were revealed by XPS analysis. Remarkable results were found even in the very low concentration of AuNPs (ratio 0.2). This AuNPs staining method is simple, cost-effective, less time consuming, and required very low concentration of AuNPs. These results can be read out through the naked eye without the help of sophisticated equipments.

Role of Surface Hydrophobicity of Dicationic Amphiphile-Stabilized Gold Nanoparticles on A549 Lung Cancer Cells

Herein, we report the surface functionality of dicationic cysteamine conjugated cholic acid (DCaC), dicationic cysteamine conjugated deoxycholic acid (DCaDC), and dicationic cysteamine conjugated lithocholic acid (DCaLC) templated gold nanoparticles (AuNPs) on mammalian cells. The haemocompatibility of the synthesized NPs was evaluated by in vitro hemolysis and erythrocyte sedimentation rate using human red blood cells (RBCs). In all of the systems, no toxicity was observed on human erythrocytes (RBCs) up to the concentration of 120 μg/mL. The anticancer activity of these dicationic amphiphile-stabilized AuNPs on A549 lung cancer cells was demonstrated by in vitro cell viability assay, intracellular reactive oxygen species estimation by DCFH-DA, apoptosis analysis using AO-EtBr fluorescence staining, DNA fragmentation analysis by agarose gel electrophoresis, and western blot analysis of caspase-3 expression. These results suggest that the cytotoxicity of AuNPs to A549 cells increase with the dose and hydrophobicity of amphiphiles and were found to be in the order: DCaLC-AuNPs > DCaDC-AuNPs > DCaC-AuNPs.

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem.

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.

An Efficient Hairy Root System for Withanolide Production in Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal is one of the most important medicinal plant belonging to the family Solanaceae. Its root have been used as a drug since ancient times, and various pharmacological effects have been attributed to the occurrence of secondary compounds like withaferin-A and withanolide-A. Recently, huge interests are generated for production of these bioactive compounds through Agrobacterium rhizogenes-mediated hairy root culture techniques. The present review explores the culture conditions for efficient Agrobacterium-mediated hairy root culture system of W. somnifera for withanolide production. The hairy root induction is influenced by several factors like bacterial stain, type of explant, and cocultivation methods. The transformation efficiency could be enhanced by the addition of acetosyringone and SAAT treatments during cocultivation. Recent studies have also shown positive correlations of elicitors and biosynthetic pathway genes on withanolide production in hairy root culture of W. somnifera.