Sarmistha Sen Raychaudhuri

The role of superoxide dismutase in combating oxidative stress in higher plants

Superoxide dismutase (SOD) isozymes are compartmentalized in higher plants and play a major role in combating oxygen radical mediated toxicity. In this review we evaluate the mode of action and effects of the SOD isoforms with respect to oxidative stress resistance, correlating age, species, and specificity of plants during development.

ENHANCED DEVELOPMENT OF SOMATIC EMBRYOS OF PLANTAGO OVATA FORSK. BY ADDITIVES

Plantago ovata Forsk (commonly known as Isabgul) is an economically important medicinal plant. In the present investigation, in vitro plant regeneration of P. ovata was attempted through somatic embryogenesis. Casein hydrolysate and coconut water were used in different concentrations in Murashige and Skoog medium along with 1-naphthaleneacetic acid and N6-benzyladenine to increase the amount of callus and number of somatic embryos. Light and scanning electron microscopic studies followed the developmental stages of embryo formation. Results indicated that optimum concentrations of casein hydrolysate and coconut water are useful for promoting the growth of embryogenic cultures. However, a supra-optimal dose of casein hydrolysate and coconut water induced polyphenol synthesis and caused browning of callus and also eventual death of embryos. The use of additives such as coconut water and casein hydrolysate promotes large-scale production of P. ovata through in vitro somatic embryogenesis.SummaryPlantago ovata Forsk (commonly known as Isabgul) is an economically important medicinal plant. In the present investigation, in vitro plant regeneration of P. ovata was attempted through somatic embryogenesis. Casein hydrolysate and coconut water were used in different concentrations in Murashige and Skoog medium along with 1-naphthaleneacetic acid and N6-benzyladenine to increase the amount of callus and number of somatic embryos. Light and scanning electron microscopic studies followed the developmental stages of embryo formation. Results indicated that optimum concentrations of casein hydrolysate and coconut water are useful for promoting the growth of embryogenic cultures. However, a supra-optimal dose of casein hydrolysate and coconut water induced polyphenol synthesis and caused browning of callus and also eventual death of embryos. The use of additives such as coconut water and casein hydrolysate promotes large-scale production of P. ovata through in vitro somatic embryogenesis.

Molecular characterization, modeling and expression analysis of a somatic embryogenesis receptor kinase (SERK) gene in Momordica charantia L. during somatic embryogenesis

Somatic embryogenesis receptor kinase (SERK) gene is known to be a marker of somatic embryogenesis in several plant species. The present study reported the isolation and characterization of a SERK gene ortholog, designated as McSERK, in Momordica charantia, an important medicinal plant. The complete coding region of McSERK was found to encode a 627 amino acid protein which contained an N-terminal signal peptide, a leucine zipper, five leucine rich repeats, a serine-proline-proline domain, a transmembrane domain, a kinase domain and the C-terminal region, depicting the typical characteristic features of SERK-family proteins. Phylogenetic analysis suggested that McSERK was highly similar to the SERK proteins of Cucumis sativus, Glycine max and Medicago truncatula. Homology modeling was attempted to construct the three dimensional structure of McSERK protein which showed that it corresponded to a monomeric protein. McSERK expression was high in embryogenic callus but its expression was relatively low in different plant organs. The high expression of McSERK transcript in the embryogenic callus confirmed its association with somatic embryogenesis in M. charantia.

Changes in esterase and superoxide dismutase isozymes during in vitro morphogenesis in Plantago ovata Forssk

Callus cultures were established from hypocotyl explants of Plantago ovata in Murashige and Skoogs medium supplemented with 2,4-d/Kinetin and NAA/BA combinations. Calluses growing on NAA/BA (0.4 mg l−1 each) regenerated into plantlets after the second subculture when transferred to media containing IAA (0.2 mg l−1) and BA (5 mg l−1). Shoot tip multiplication was carried out in the same media with IAA and BA. Tissue samples from calluses, regenerating plantlets and multiplying shoot tips grown in vitro were extracted with protein extraction buffer and subjected to esterase and superoxide dismutase isozyme analysis. The calluses however, showed a uniform banding in esterase even when grown on different hormone combinations. The multiplying shootlets showed two new bands which were not found in either the control or the regenerating plants. A new band was also found in the multiplying shootlets when analysed for superoxide dismutase. It is postulated that those new enzyme forms which arise in esterase as well as in superoxide dismutase may either arise de novo or due to post-transcriptional modification of the genes and are essential for shoot tip multiplication of Plantago ovata.

Peroxidase changes in barley induced by ionizing and thermal radiation

Thermal and ionizing (gamma-ray) radiations were used to induce damage to barley seeds (IB65). The activity and isozyme banding patterns of peroxidase were compared. It was found that both physical agents caused damage to barley seeds (as observed from seedling height), but their action on peroxidase activity is not similar. Gamma-Rays enhance peroxidase activity. Thermal radiation, on the other hand, tends to reduce it but fails to alter the number of peroxidase isozymes. It is conjectured that the pathways of damage by thermal and ionizing radiations are not the same.

Radiation-induced phenotypic alterations in relation to isozymes and RAPD markers in Vigna radiata (L.) Wilczek

Purpose: The present investigation is aimed at studies on the effects of gamma rays on in vitro and in vivo damage in Vigna radiata. The parameters studied are germination frequency, seedling injury, isozyme alteration and random amplified polymorphic DNA (RAPD) markers. Results obtained are analyzed in the light of modern applications of radiation damage. Materials and methods: Seeds of Vigna radiata were subjected to gamma irradiation with a dose of 20 – 200 Gy. The percent of seedling damage and frequency of germination were determined. Callus samples were produced in vitro and exposed to gamma rays. The irradiated callus samples were processed to extract total protein, and specifically stained for superoxide dismutase (SOD) and peroxidase isozymes. Total genomic DNA was extracted from irradiated callus samples and subjected to random amplified polymorphic DNA analysis using 23 random decamer primers. Results: Gamma irradiation resulted in retardation in seedling height and decrease in germination frequency in a dose dependent manner. Inhibition assay identified variation in response between different isoforms of SOD on radiation exposure. Changes in peroxidase activity were also observed following irradiation. RAPD analysis showed that new bands appeared in the 20 Gy irradiated sample which in the case of some primers showed similarity with the control. The calli irradiated with 50 Gy and 100 Gy of gamma rays was found to have striking resemblance in banding pattern. Callus irradiated at 200 Gy showed maximum damage. DNA damage as revealed by RAPD analysis was reflected in the appearance of new bands with varying molecular weights. Conclusion: New isoforms of SOD appeared after irradiation followed by 24 h recovery. Some isoforms of peroxidase reappeared in calli after 24 h recovery. Results of RAPD analysis indicated that the DNA polymorphism was dose dependent.

PIXE analysis of trace elements in relation to chlorophyll concentration in Plantago ovata Forsk

Plantago ovata Forsk - an economically important medicinal plant - was analyzed for trace elements and chlorophyll in a study of the effects of gamma radiation on physiological responses of the seedlings. Proton-induced X-ray emission (PIXE) technique was used to quantify trace elements in unirradiated and gamma-irradiated plants at the seedling stage. The experiments revealed radiation-induced changes in the trace element and chlorophyll concentrations.

Antioxidant activity and high-performance liquid chromatographic analysis of phenolic compounds during in vitro callus culture of Plantago ovata Forsk. and effect of exogenous additives on accumulation of phenolic compounds.

BACKGROUND Plantago ovata, commonly called psyllium, is known to be a rich source of polyphenolic compounds. The present study was aimed at determining polyphenol content and studying their antioxidant activities in P. ovata during in vitro callus culture. An attempt was also made to enhance polyphenol content using external additives. The role of PAL gene in polyphenol accumulation was also studied. RESULTS The study indicated the presence of significant amounts of polyphenols, including flavonoids, in P. ovata callus. A gradual increase in polyphenol and flavonoid content was observed up to the third passage (63 days) of callus culture, which declined at the next passage. The third-passage callus showed highest antioxidant activity. High-performance liquid chromatographic results indicated the presence of high amounts of gallic acid and rutin in P. ovata calli; however, other polyphenols were also present but to a lesser extent. Additive supplementation was effective in enhancing polyphenol production and in increasing antioxidant activity in P. ovata callus. CONCLUSION The present research reported accumulation of polyphenols in callus culture of P. ovata, which could be applied to isolation of polyphenols for various beneficial purposes. It also indicated enhancement in the production of several polyphenols and also an increase in antioxidant activity in the additive-treated callus.

In Vitro Radiation Induced Alterations in Heavy Metals and Metallothionein content in Plantago ovata Forsk

Proton Induced X-ray emission (PIXE) and fluorescence-activated cell sorting (FACS) have been used to study the effects of gamma irradiation on heavy metal accumulation in callus tissue of Plantago ovata—an important cash crop of India. PIXE analysis revealed radiation-induced alteration in trace element profile during developmental stages of the callus of P. ovata. Subsequent experiments showed antagonism between Fe and Cu and also Cu and Zn and synergistic effect between Fe and Zn. FACS analysis showed significant induction of the metallothionein (MT) protein following gamma-irradiation, and maximum induction was noted at the 50-Gy absorbed dose. This indicated a progressive increment of MTs as a measure for protection against gamma-rays, to combat alteration in the homeostasis of heavy metals like Fe, Cu, Zn, and Mn.

Radiation induced alterations in Vigna radiata during in vitro somatic embryogenesis.

Purpose: Tissue culture has been exploited to understand molecular aspects of regeneration potential of the plants in normal and in stressed conditions. The present study describes ionizing radiation from 60Co source as the stress stimulator to assess in vitro development of somatic embryo of Vigna radiata, a protein-rich pulse. Materials and methods: Callus culture was established, using leaves of V. radiata. Somatic embryogenesis was induced by manipulating plant hormones. Calli were exposed to gamma rays. Genomic DNA isolated from gamma-irradiated callus samples were subjected to random amplified polymorphic DNA analysis. A band of molecular weight 1440 bp was used as a probe and Southern hybridization was carried out. To determine alterations in DNA following irradiation, RAPD bands were cloned and sequenced from control and irradiated samples. Embryogenic calli were exposed to gamma irradiation and the effects were assessed immediately and after seven days of exposure. Phenotypic alterations were observed using scanning electron microscopy. Results: Exposed calli revealed altered frequency of somatic embryo formation. Results showed that the 1440 bp molecular weight probe hybridized with bands of low molecular weight. DNA sequences from irradiated samples showed recombination when compared to control. Scanning electron micrography illustrated presence of transient pores on the exposed embryos. BLAST search of the DNA sequences showed partial homology with some sequences from Arabidopsis thaliana. Conclusion: The present report might help in designing a breeding program, where both radiation coupled with somatic embryogenesis could be employed to build up the desired variants.