Kavindra Nath Tiwari

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field.

Micropropagation of Centella asiatica (L.), a valuable medicinal herb

A protocol is described for rapid and large-scale in vitro clonal propagation of the valuable medicinal herb Centella asiatica (L.) by enhanced axillary bud proliferation in nodal segments isolated from mature plants. Although bud break was dependent on BA supply, the synergistic combination of 22.2 μM BA and 2.68 μM NAA induced the optimum frequency (91%) of shoot formation as well as shoot number (4 to 5 shoots per node). Subculturing of nodal segments harvested from the in vitro derived axenic shoots on the multiplication medium enabled continuous production of healthy shoots with similar frequency. MS medium supplemented with 6.7 μM BA and 2.88 μM IAA was found most suitable for shoot elongation. Rooting was highest (90%) on full-strength MS medium containing 2.46 μM IBA. Micropropagated plants established in garden soil were uniform and identical to the donor plant with respect to growth characteristics. This micropropagation procedure could be useful for raising a stock of genetically homogenous plant material for field cultivation.

Enhancement of Nodulation and Yield of Chickpea by Co-inoculation of Indigenous Mesorhizobium spp. and Plant Growth–Promoting Rhizobacteria in Eastern Uttar Pradesh

We study the effect of plant growth–promoting rhizobacteria (PGPR) along with Mesorhizobium sp. BHURC02 on nodulation, plant growth, yield, and nutrient content of chickpea (Cicer arietinum L.) under field conditions. A similar study has been conducted for nodulation and plant growth of chickpea in pot experiment under glasshouse conditions. The treatment combination of Mesorhizobium sp. BHURC02 and Pseudomonas fluorescens BHUPSB06 statistically significantly increased nodule number plant–1, dry weight of nodule plant–1, and root and shoot dry weights plant–1 over the control under a glasshouse experiment. The maximum significant increase in nodule number, dry matter, and nutrient content were recorded in co-inoculation of Mesorhizobium sp. BHURC02 and P. fluorescens BHUPSB06 followed by co-inoculation of Mesorhizobium sp., Azotobacter chroococcum, and Bacillus megatrium BHUPSB14 over uninoculated control in a 2-year field study. Hence, co-inoculation of Mesorhizobium sp. and P. fluorescens may be effective indigenous PGPR for chickpea production.

Shoot regeneration from immature cotyledons of Cicer arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm−3 zeatin with or without 0.04 mg dm−3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 − 1 mg dm−3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm−3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA3). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm−3 IBA for 5 min and culture on growth regulator free half-strength MS medium.

Antioxidant Property of Aerial Parts and Root of Phyllanthus fraternus Webster, an Important Medicinal Plant

In present study free radical scavenging potential of aerial parts and root of Phyllanthus fraternus was investigated. Extraction was done in water and ethanol. Total antioxidant capacity was measured by DPPH free radical scavenging method; ethanolic extract of aerial part was most potent in activity with 50% inhibition at 258 μg/mL concentration. Lipid peroxidation (LPO) was measured in terms of thiobarbituric acid-reactive substances (TBARS) by using egg-yolk homogenates as lipid-rich media with EC50 of aerial part (ethanolic) 1522 μg/mL which was found to be most active. Superoxide (SO) radical scavenging activity was measured using riboflavin-light-nitroblue tetrazolium assay. Ethanolic and aqueous extract of both aerial part and root was almost similar in superoxide radical scavenging activity. Reducing power was determined on the basis of Fe3+-Fe2+ transformation in the presence of extract. Total phenolic and flavonoid contents were also measured by spectroscopic method. Results showed that the ethanolic fraction of aerial part is most active towards antioxidant potential and this activity is related to its polyphenolic content and reducing potential. Thus, P. fraternus extract can be used as potent natural antioxidant.

Comparative Antioxidant Study of Stem and Stem Induced Callus of Phyllanthus fraternus Webster—An Important Antiviral and Hepatoprotective Plant

Phyllanthus fraternus is widely used in the cure of various liver diseases and possess antiviral properties especially against hepatitis virus. In the present study, evaluation of the antioxidant activity of stem and calli induced from stem has been done by different assays. Extraction was done by standard method in water and ethanol. Total antioxidant capacity was measured by 1, 1-diphenyl-2-picrylhydrazyl free radical scavenging method. Lipid peroxidation was measured in terms of thiobarbituric acid-reactive substances (TBARS) by using egg yolk homogenates as lipid-rich media, and superoxide radical scavenging activity was measured using riboflavin–light–nitro blue tetrazolium assay. Reducing power was determined on the basis of Fe3+–Fe2+ transformation in the presence of the extract. In addition to the antioxidant activity, polyphenolic compounds like total phenolics and flavonoids were also measured by spectroscopic method. Results showed that the ethanolic extract of stem is more potent in antioxidant activity than its aqueous extract and ethanolic extract of calli. A significant correlation between antioxidant capacity and polyphenolic content and reducing potential was observed, indicating that phenolic compounds and reducers present in extract are major contributors to the antioxidant potential. Thus, this plant extract could be used as a potent natural antioxidant.

Synergistic effect of trimethoprim and bavistin for micropropagation of Bacopa monniera

A micropropagation protocol for Bacopa monniera (L.) Wettst., a medicinally important plant, has been developed. Direct organogenesis without callus formation was induced by culturing node, internode and leaf explants on growth regulator free Murashige and Skoog (MS) medium. MS medium supplemented with an antibiotic trimethoprim (TMP) and a fungicide bavistin (BVN) produced axillary shoots from node and adventitious shoot buds on the surface of all explants. The combination of 200 mg dm−3 TMP and 200 mg dm−3 BVN induced the optimum frequency of shoot formation as well as shoot number. Presence of both TMP and BVN induced multiple axillary shoot formation from the nodal segments and this ability was maintained for four subcultures.

Premna integrifolia ameliorates cyclophosphamide-induced hepatotoxicity by modulation of oxidative stress and apoptosis

The present study was designed to evaluate the ameliorative effect of ethyl acetate extract of Premna integrifolia L. (EAEPI) leaves in cyclophosphamide (CP)-induced hepatic injury in mice. Mice were intoxicated with CP (200 mg/kg b. wt., i.p.) for 5 weeks or EAEPI (400 and 600 mg/kg b. wt., orally) in combination with CP. The results demonstrated that EAEPI exerts protective effect against CP induced hepatotoxicity, as evident from restoration of altered haematological parameters and alleviations of liver marker enzymes in serum. EAEPI also attenuated oxidative stress and antioxidant markers as evident from reversal of lipid peroxidation, glutathione levels as well as activities of catalase and superoxide dismutase enzymes. Moreover, EAEPI attenuated apoptosis and histopathological liver tissue damage in CP-intoxicated mice. In conclusion, EAEPI could protect mice liver against cyclophosphamide toxicity by inhibiting oxidative stress and apoptosis.The protective activity of EAEPI may be due to presence of polyphenolic compounds as identified by UHPLC-Q-TOF-MS/MS.

Assessment of factors on shoot proliferation potential of nodal explants of Phyllanthus fraternus and assessment of genetic fidelity of micropropagated plants using RAPD marker

Phyllanthus fraternus is an important medicinal plant, popularly known for its hepatoprotective and antiviral activities since ancient times. Various physiological factors like carbon sources, concentration of agar, pH of the media and effect of season of explants collection were optimized for high frequency regeneration of P. fraternus. The frequency of regeneration, average number and length of shoots were highly influenced by the type and concentration of carbon sources (monosaccharides and disaccharides, 1 to 4%), agar concentration (0.2 to 1%) and pH (4.5 to 6.8) of the media. Media containing 3% sucrose, 0.6% agar at pH 5.8 was best for regeneration. Seasonal variation of explants collection significantly affected the axillary shoots proliferation from explants and best proliferation was observed from explants collected during April to June. Genetic fidelity of regenerated plants was assessed by random amplified polymorphic DNA markers. No polymorphism was detected in micropropagated plants and the mother plant, revealing the genetic homogeneity of the in vitro raised plantlets. This is the first report regarding establishment of genetic fidelity of micropropagated P. fraternus plants, which could be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses.