Indian Journal of Biotechnology




APRIL 2002






Calcium homeostasis in plants: role of calcium binding proteins in abiotic stress tolerance.



  Giridhar Pandey, M K Reddy, Sudhir K Sopory & Sneh lata Singla-Pareek






New curcumin – bioconjugate: synthesis and DNA binding.


  Sanjay Kumar, Vibha Shukla, Arvind Misra, Snehlata Tripathi & Krishna Misra




Cloning and characterization of a gene encoding ubiquitin conjugating enzyme from the mangrove species, Avicennia marina (Forsk.) Vierh.



  M Parani, M N Jithesh, M Lakshmi & A Parida




Antigenic differentiation of Equine Herpes Virus -1 (EHV-1) isolates of Indian origin using monoclonal antibodies.



  B K Singh, B R Gulati, S C Tewari & M P Yadav




Viral etiology of Complete Hydatidiform Moles (CHM)


  Swapna Alex, Beena Panikkar, P K Shyamala & Prabha Balaram




Efficacy of a rock phosphate based soil implant formulation of phosphobacteria in soybean (Glycine max Merrill).



  G Viveganandan & K S Jauhri




Role of disulfide bridges in structure-activity relationship of plant lipases (from wheat germ and rice bran).



  K N Gopalakrishna, Purnima Kaul, P Ramesh Kumar & V Prakash




Micropropagation of Salvadora persica Linn. via cotyledonary nodes.


  Sujata Mathur, Gyan Singh Shekhawat & Amla Batra




A technique for rapid micropropagation of Solanum surattense Burm. f.


  P K Pawar, C S Pawar, B A Narkhede, N P Teli, S R Bhalsing &
V L Maheshwari




Short Communications


Mass cultivation of Bacillus thuringiensis var. kurstaki in fed-batch culture for high spore count and improved insecticidal activity.



  Vinod Bihari, C K M Tripathi, Banani Sur, W M Liu & Rakesh Bajpai








Ethanol-induced enhancement of the transformation of Escherichia coli by plasmid DNA.



Suchitra Sarkar, Sujan Chaudhuri & Tarakdas Basu




Introduction of Gerbera cultivation in Lucknow agro-climate through tissue culture of young flower head.



  A K A Mandal & S K Datta




News Scan


Tissue engineering


  K Y Kavathekar




Instructions to Contributors











Calcium Homeostasis in Plants: Role of Calcium Binding Proteins in Abiotic Stress Tolerance

Giridhar Pandey  M K Reddy, Sudhir K Sopory and Sneh lata Singla-Pareek


A majority of environmental signals of varied types as well as varied intensities are perceived at the membrane level in a cell. In case of multicellular organisms like plants or animals, this ‘perception’ not only needs to be transferred to the actual centre of controlling and responding unit i.e. the nucleus but sometimes from one cell to another cell which may just be lying close enough or even at an appreciable distance e.g. ‘root-shoot communications’. In contrast to processes of cell-to-cell communication in a plant system, which is just beginning to be elucidated, the mechanism of ‘transfer’ of this information from outer surface to the core controlling units has been an active area of research since past few decades. Abundant reports do exit in literature, which support a kind of ‘cascading mechanism’ for this purpose. It is now a well-established fact that a divalent cation i.e. Ca2+ plays an extremely important role in this process. The fluctuations in the level of Ca2+ at a given time in a given cell organelle is the crucial factor determining the activation stage of some special proteins, which have been proposed to have a high affinity for Ca2+. Such proteins are known as Ca2+ -binding proteins (CaBPs). Under environmental abuses, the level and activation of CaBPs play an important role in bringing about the ‘ignition’ of ‘protective’ as well as ‘defensive’ mechanisms, which ultimately are reflected in the form of the physiological adaptations in the plant as a whole system. The present review is an attempt to highlight the importance of CaBPs in plants under abiotic stress conditions.

Keywords: abiotic stresses, adaptations, Ca2+, CaBPs, signal transduction, stress tolerance, transgenic plants



New Curcumin – Bioconjugate: Synthesis and DNA Binding


Sanjay Kumar, Vibha Shukla, Arvind Misra, Snehlata Tripathi and Krishna Misra*


A triglycyl derivative of curcumin, 1,7-bis (4-O-glycinoyl-3-methoxy phenyl)-1,6-heptadine-C-4-glycinoyl-3, 5-dione, was synthesized and characterized by UV, elemental analysis and 1H NMR. Interaction studies of curcumin and curcumin-bioconjugate with calf thymus DNA were carried out using UV-absorbance, gel electrophoresis and viscometric studies. Curcumin-bioconjugate was found to be A-T specific minor groove binder.

Keywords: curcumin, glycine, bioconjugate, viscosity, interaction, groove binding


Cloning and Characterization of a Gene Encoding Ubiquitin Conjugating Enzyme from the Mangrove Species, Avicennia marina (Forsk.) Vierh.

M Parani, M N Jithesh, M Lakshmi and A Parida


Covalent attachment of ubiquitin has been implicated in mediating proteolysis of the cellular proteins by Ubiquitin–proteasome pathway. Ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3) are the three enzymes involved in this process. This paper reports the isolation of a gene that codes for the ubiquitin conjugating enzyme in Avicennia marina (AmUBC2), and regulation of its expression at RNA level under salt stress. Deduced amino acid sequence of AmUBC2 showed 96% identity with UBC2 of Arabidopsis thaliana and also 73-78% identity with RAD6 DNA repair protein of Homo sapiens, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Multiple alignment analysis showed that the amino acid residues in the core region of UBC2 were highly conserved across different taxa in the evolutionary hierarchy. While some ubiquitin conjugating enzymes were induced under salt, heat and heavy metal stress in different tissues in plants, Northern analysis in the present study has clearly shown that the expression of UBC2 is not induced by salt stress either in root or in leaf tissues in A. marina. Southern hybridization of genomic DNA with gene-specific probe showed that AmUBC2 is a single copy gene.

Keywords: ubiquitin conjugating enzyme, Avicennia marina, proteolysis, mangroves



Antigenic Differentiation of Equine Herpes Virus-1 (EHV-1) Isolates of Indian Origin using Monoclonal Antibodies


B K Singh, B R Gulati, S C Tewari and M P Yadav


A panel of seven monoclonal antibodies (Mabs) (5 non-neutralizing and 2 neutralizing) raised against 140 kDa polypeptide of EHV-1 strain (Hisar-90-7) were used for antigenic characterization of five Indian isolates and compared with reference strain (592) of EHV-1. Jind-96 and Hisar-90-7 were antigenically indistinguishable from reference strain in indirect enzyme-linked immunosorbent assay (ELISA) using 5 non-neutralizing Mabs. The antigenic differences were observed in three EHV-1 isolates i.e. Tohana-96-2, Delhi-98 and Raj-98 as they failed to react with 5, 4, and 1 of five non-neutralizing Mabs, respectively. While all the six virus isolates were neutralized with Mab 9C4 in virus neutralization test (VNT), two (Tohana-96-2 and Raj-98) of the 6 viral isolates were not neutralized with one Mab, 1H6. However, EHV-1 isolates could not be differentiated on western blot analysis as all the Mabs reacted with a 140 kDa protein in these isolates. The findings of the ELISA and VNT using 7 Mabs indicate that initial isolates (Hisar-90-7 and Jind-96) are antigenically closer to reference strain (592) and there is emergence of antigenically different isolates (Tohana-96-2, Delhi-98 and Raj-98) subsequently. The study also establishes that more than one antigenically different strains of EHV-1 are circulating in equines of northern India.

Keywords: equine herpes virus-1, monoclonal antibodies, antigenic differentiation, ELISA, virus neutralization, western blotting





Viral Etiology of Complete Hydatidiform Moles (CHM)

Swapna Alex, Beena Panikkar, P K Shyamala and Prabha Balaram


Complete hydatidiform moles (CHM) are the most common forms of gestational trophoblastic disease. CHM prevalence rate is higher in Kerala, India as compared to other parts of the world. The etiology of this disease is not yet clearly understood. Reports and observations suggest conceptual alterations, which could be due to involvement of viruses or carcinogens rather than host factors. In this study, the association of the common genital viruses with this disease was examined in 105 cases of CHM and 95 cases of normal placentae using immunohistochemistry, ELISA and PCR techniques. Present study suggests an association of human papilloma virus (HPV) infection with this disease while no association was noticed with other genital viruses such as Human immunodeficiency virus (HIV), Cytomegalovirus (CMV) and Herpes simplex virus (HSV). This is the second report in world literature and first in Indian literature showing such an association.

Keywords: CHM, HIV, HPV, HSV, CMV


Efficacy of a Rock Phosphate Based Soil Implant Formulation of Phosphobacteria in Soybean (Glycine max Merrill)


G Viveganandan and K S Jauhri


For improving the effectiveness of low-grade rock phosphate, a granular formulation was developed by immobilizing phosphate solubilizing bacteria (PSB) impregnated rock phosphate in calcium alginate. This process ensured requisite standards of PSB viability in rock phosphate. The formulation  was compared with powdered soil and seed formulations for its efficacy in soybean; maximum weight of nodule, dry matter and grain yield, N and P uptake of shoot and grain were recorded with granular preparation. The granular formulation can directly be applied in soil and is environmentally safe.

Keywords: rock phosphate, phosphobacteria, formulation, efficacy, soybean


Role of Disulfide Bridges in Structure-Activity Relationship of Plant Lipases from Wheat germ and Rice bran


K N Gopalakrishna, Purnima Kaul, P Ramesh Kumar and V Prakash


Disulfide bonds of wheat germ lipase (WGL) and rice bran lipase (RBL) were reduced with sodium borohydride (NaBH4) and dithiothreitol (DTT) under non-denaturing conditions to assess the activity, conformation and stability of reduced form with those of native enzyme by using kinetics, far UV-CD, fluorescence, thermal denaturation studies and microcalorimetric measurements. Activity of reduced lipases was found to decrease in a sequential manner involving atleast two steps in both the lipases. The CD spectrum in the far UV-region indicates that overall conformation was drastically affected upon reduction. A decrease in tryptophanyl fluorescence was observed without any shift in the emission maximum. The apparent thermal denaturation temperature [Tm (app)] of the reduced WGL and RBL was lowered by 6°C and 12°C, respectively, from the native enzyme. Reduction and carboxymethylation of all four cysteines caused extensive unfolding of the enzymes resulting in the loss of activity, conformation and thermal stability significantly indicating that the disulfide bonds have a major role in stabilizing the native conformation and stability of these two lipases.

Keywords: lipase; wheat germ; rice bran; disulfide bond; activity; conformational stability; thermal stability.




Micropropagation of Salvadora persica Linn. via Cotyledonary Nodes

Sujata Mathur, Gyan Singh Shekhawat and Amla Batra


An efficient and reliable protocol for micropropagation of Salvadora persica Linn. has been standardized, which is a medicinally as well as economically important arid zone plant species. Cotyledonary nodes (1cm long) excised from 15-20 days-old seedlings germinated in vitro served as explant source. The seeds were germinated on half strength MS medium devoid of phytohormones. Cotyledonary nodes were cultured on MS medium supplemented with different concentrations of cytokinins (BAP and KN) and auxins (IAA, IBA and NAA). Maximum shoot proliferation from single explant was obtained on MS medium incorporated with BAP (4.0 mg/l), IAA (0.5 mg/l), adenine sulphate (40 mg/l), glutamine (100 mg/l) and thiamine HCl (10 mg/l). In vitro produced shoots were induced to root on a range of IBA concentrations (0.5-5.0 mg/l) supplemented to half strength MS medium. The highest frequency of root proliferation was on half strength MS medium supplemented with 3.0 mg/l IBA. The regenerates were transferred to field conditions after acclimatization with a success rate of 60%.

Keywords : agro-forestry, cotyledonary node, micropropagation, miswak, Salvadora persica


A Technique for Rapid Micropropagation of Solanum surattense Burm. f.


P K Pawar, C S Pawar, B A Narkhede, N P Teli, S R Bhalsing and V L Maheshwari

In Solanum surattense, a medicinally important member of family Solanaceae, rapid micropropagation and direct organogenesis was achieved from shoot tip and leaf explants, respectively. Both leaf and shoot tips differentiated into number of small shoots without apparently forming any callus. Average 180-200 adventitious shoots were produced from a single explant on MS basal medium fortified with BAP and kinetin individually and in combination. Caulogenesis was followed by rhizogenesis on the same medium without phytohormone. In vitro regenerated plantlets were healthy with dark green leaves and attained a length of 5-10 cm in 10 weeks. The post- transplantation survival rate in the field was 80±5% without any seasonal constraints.

Keywords: leaf culture, shoot tip culture, Solanum surattense, solasodine





Mass Cultivation of Bacillus thuringiensis var. kurstaki in Fed-Batch Culture for High Spore Count and ImprovedInsecticidal Activity


Vinod Bihari, C K M Tripathi, Banani Sur, W M Liu and Rakesh Bajpai


 Batch and fed-batch cultivation of Bacillus thuringiensis var. kurstaki was carried out to achieve high cell and spore density with maximum insecticidal activity. Cell cultivation was done in fully equipped fermentors under controlled conditions at different feed rates. Increased cell mass, high spore counts and improved insecticidal activity were achieved with fed-batch cultivation system. With the appropriate supply of glucose and other nutrients, it was possible to grow the cells to >50 g (dry weight)/l density and achieve a spore concentration of 3.9x1010/ml having high insecticidal activity.

Keywords: insecticidal activity, fed-batch fermentation, sporulation, crystal potency, glucose feeding



Ethanol-induced Enhancement of theTransformation of Escherichia coli by


Suchitra Sarkar, Sujan Chaudhuri and Tarakdas Basu


 The transformation efficiency (TR)E of CaCl2-treated competent E. coli cells by plasmid DNA decreased gradually by the presence of increasing ethanol concentration (up to 10% v/v) in the cell population during transformation. This was due to ethanol-induced leaching of lipopolysaccharide (LPS) from the competent cell surface. On the other hand, when the ethanol was removed after treatment of the competent cells for 30 min and then the cells were allowed to transform, the (TR)E enhanced compared to that of control cells; the enhancement was found to be maximum (> 100%) by the treatment of the cells with 5% (v/v) ethanol.

Keywords: ethanol, E. coli, calcium chloride, plasmid DNA, LPS, transformation efficiency



Introduction of Gerbera Cultivation in Lucknow Agro-climate through Tissue Culture of Young Flower Head


A K A Mandal and S K Datta

Gerbera (Gerbera jamesonii H. Bolus ex Hook. f.) a temperate crop, distributed in the temperate Himalayas from Kashmir to Nepal at altitudes of 13,00 to 32,00 m. It is very difficult to grow this temperate crop in sub-tropical climate of Lucknow. Organogenic cultures were established from immature flower heads on modified MS medium supplemented with IAA and BA. Regenerated shoots were rooted on growth regulator-free medium. Rooted shoots were hardened during July-August and transferred to field conditions, where they grew well and produced flowers within 6-8 months.

Keywords: Gerbera introduction, shoot regeneration, sub-tropical climate