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Indian Journal of Biotechnology

 

 

 

VOLUME 2

NUMBER 4

OCTOBER 2003

 

 

CONTENTS

 

Reviews

 

Evaluation of yeast as an expression system

477

M W Nasser, V Pooja, M Z Abdin and S K Jain

 

 

 

Recent advances in vaccinology

494

Y Udaya Bhaskara Rao

 

Papers

 

Emergence of antibiotic resistance in nosocomial strains of coagulase negative staphylococci (CNS)

 

499

M S Geetha, C Ashok and S B Sullia

 

 

 

Development of long-term cell suspension cultures of wild tomato species, Lycopersicon chilense Dun. as regular source of protoplast: An efficient protoplast-to-plant system

 

 

504

R S Patil, M R Davey, J B Power and E C Cocking

 

 

 

Comparison of Lycopersicon peruvianum Mill. and L. chilense Dun. for development of tomato somatic hybrids

 

512

R S Patil, M R Davey, J B Power and E C Cocking

 

 

 

Agrobacterium tumefaciens-mediated transformation of chickpea (Cicer arietinum L.) using mature embryonic axes and cotyledonary nodes

 

524

Indraneel Sanyal, A K Singh and D V Amla

 

 

 

Expression of capsid precursor polypeptide (P1) of foot and mouth disease virus vaccine strains in Pichia pastoris

 

533

R Renji, V Balamurugan, S N Saha, G R Reddy and V V S Suryanarayana

 

 

 

Comparison of insecticide resistant and susceptible populations of Spodoptera litura Fab.

539

S Janarthanan, S Seshadri, K Kathiravan and S Ignacimuthu

 

 

 

Induced immunity against haemolymph proteins of Anopheles stephensi: Effects of fecundity and transmission blocking of malaria parasite

 

543

Monika Gulia and S K Gakhar

 

 

 

Expression of recombinant fatty acid binding protein from Fasciola gigantica

549

D Sriveny, O K Raina, Jaya Verma and S C Yadav

 

 

 

Protein polymorphisms in mastomys [Praomys (mastomys) coucha]

553

B Maity, N Gupta and P Y Guru

 

 

 

Nitrogen stress induces Magnaporthe grisea (Herbert) Barr to secrete protoplast-disrupting proteins

 

558

R Rathour, B M Singh and P Plaha

 

 

 

Optimization of medium and cultural conditions for neomycin production using response surface methodology

 

564

K Adinarayana, P Ellaiah, B Srinivasulu, J Lakshmi Narayana and K V V S N Bapi Raju

 

 

 

Studies on hydantoinase production using Pseudomonas strain

571

B G Gaikwad, S Nene and B D Kulkarni

 

 

 

Use of heavy metals for quantification of rhizobia and suppression of bacterial contaminants in carrier based inoculants

 

577

R P Jeevan Kumar and K S Jauhri

 

 

 

Isolation and characterization of aluminium and copper resistant 'P' solubilizing alkalophilic bacteria

 

583

Shilpi Mittal, Jean-Marie Meyer and Reeta Goel

 

 

 

Evaluation of multiplex reverse transcription polymerase chain reaction (RT-PCR) for simultaneous detection of potato viruses and strains

 

587

Yogita Verma, Shivali Sood, Y S Ahlawat, S M Paul Khurana, X Nie and R P Singh

 

 

 

Plant defence activators inducing systemic resistance in Zingiber officinale Rosc. against Pythium aphanidermatum (Edson) Fitz.

 

591

N C Karmakar, Rajyasri Ghosh and R P Purkayastha

 

 

 

Caspase-3 like protein in wheat-Tilletia indica dual culture system as potential biomarker for host resistance to Karnal bunt

 

596

Niroj Kumar Sethy, Dinesh Pandey, U S Singh and Anil Kumar

 

 

 

Design and optimization of novel immuno-dipstick test for detection of teliospores of Karnal bunt (Tilletia indica) of wheat

 

603

Ravi Kesri and Anil Kumar

 

 

 

Short Communications

 

 

 

Strain differentiation of Salmonella Bareilly by AP-PCR

607

M K Saxena, V P Singh, Anjani Saxena, B D Lakhchura and B Sharma

 

 

 

Biotransformation of ferulic acid to vanillin by locally isolated bacterial cultures

610

Renu Agrawal, Y N Seetharam, R Chandrakanth Kelamani and G Jyothishwaran

 

 

 

High-rate vermicomposting systems for recycling paper waste

613

S Gajalakshmi and S A Abbasi

 

 

 

News Scan

 

Yeast made somewhat human to produce human proteins

New insights into DNA transcription

 

616

 

 

List of Referees

618

 

 

Annual Author Index

619

 

 

Annual Keyword Index

621

 

 

Instructions to Contributors

625

 

 

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 477-493

 

Evaluation of Yeast as an Expression System

M W Nasser, V Pooja, M Z Abdin and S K Jain*

 

Developments in recombinant DNA technology have provided an alternate route for the production of proteins. Gene expression and production of proteins of interest are of great importance for basic research as well as for biomedical applications. A number of expression systems using mammalian cells, insect cells, yeast and other bacteria as host have been developed. Yeast has received attention as a suitable host for expression of many mammalian genes due to many specific characteristics. Yeast strains, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris, have many advantages over other systems and may be the host of choice for the expression of complex proteins of therapeutic value. During the post-genomic era, the importance of these strains for the expression of heterologous genes may enhance considerably.

 

Keywords: Schizosaccharomyces pombe, Pichia pastoris, gene expression, glycosylation, secretion

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 494-498

 

 

Recent Advances in Vaccinology

Y Udaya Bhaskara Rao*

 

Vaccination continues to be the most cost effective, safest and most powerful tool of medicine for preventing sufferings, disability and death from infectious diseases. The traditional vaccines were developed when one knew very little about how the diseases were caused and how the immunity worked. Most of the vaccines were developed by trial and error in animal and human experiments. With the recent advances in molecular biology, immunology and recombinant DNA technology, one can understand how the antigens are processed and presented to the immune system and how that affects immune response. Also the current researches are underway into the mechanism of use of pathogens to infect and cause disease. The developments in vaccinology with DNA vacinnes, transgenic plant vaccines and combination of vaccines promises an exciting area in prevention and control of infectious diseases.

 

Keywords: vaccination, DNA vaccines, immunization, immune response, cytotoxic T cells

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 499-503

 

 

Emergence of Antibiotic Resistance in Nosocomial Strains of Coagulase Negative Staphylococci (CNS)

M S Geetha, C Ashok and S B Sullia*

 

Nosocomial infections caused by Coagulase Negative Staphylococci (CNS) have reached epidemic proportions. In the present work, about 26 clinically significant CNS belonging to Staphylococcus epidermidis and S. saprophyticus, were isolated from various clinical specimens and hospital environments. A few CNS strains have shown multiple antibiotic resistance to ampicillin, cotrimoxazole, cephalexin, cefotaxime, penicillin, tetracycline, gentamicin and lincomycin. Plasmid analysis of representative clinical isolates demonstrated the presence of 2-29.9 kb sized plasmid DNA. Intra and inter-specific gene transfer was observed between different species of CNS and S. aureus which suggests the rapid emergence of multiple antibiotic resistance in CNS.

 

Keywords: Coagulase Negative Staphylococci (CNS), antibiotic resistance, plasmid DNA

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 504-511

 

 

Development of Long-term Cell Suspension Cultures of Wild Tomato Species, Lycopersicon chilense Dun. as Regular Source of Protoplast: An Efficient Protoplast-to-Plant SystemR

R S Patil*, M R Davey, J B Power2 and E C Cocking

 

 

Initiation of direct and indirect cell suspension cultures of L. chilense Dun. was confined to the origin of auxin in the culture medium. Direct cell suspensions were initiated in MSP1 culture medium containing NAA while indirect suspensions were initiated in culture media UM and UMI containing 2,4-D. The growth of direct and indirect cell suspensions was distinguished by the growth rate and phenolic production. Direct suspensions have shown a continuous growth in growth curve studies (8 day) and recorded a significantly higher cell growth in comparison to two other indirect cell suspensions. In indirect suspensions, 2,4-D toxicity arose after 5 months of culture and as a consequence loss of vigour followed by phenolic browning occurred. Direct cell suspensions proved to be long-term cultures without any adverse effects of NAA on the growth rate and were genetically stable. Higher protoplast yields were obtained from the direct cell suspension cultures which were associated with the increased growth rate. A productive protoplast-to-plant system was developed.

 

Keywords: Lycopersicon chilense, cell suspension cultures, protoplasts, plant regeneration

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp.512-523

 

 

Comparison of Lycopersicon peruvianum Mill. and L. chilense  Dun. for Developement of Tomato Somatic HybridsR

R S Patil*, M R Davey, J B Power and E C Cocking

 

Lycopersicon peruvianum Mill. and L. chilense Dun., two species of ‘Peruvianum complex’ were compared for production and characterisation of tomato somatic hybrids. The double selection system “employing kanamycin resistance from tomato (L. esculentum Mill.) cultivar, Pusa Ruby and protoplast regeneration pathway from wild tomato parents” was used for identification of heterokaryons. Somatic hybrid plants were characterised by morphological markers, cytogenetic studies and biochemical analyses. The molecular confirmation of tomato somatic hybrids was obtained by PCR-based DNA markers. The importance of selectable marker (nptII) gene from cultivated species during characterisation of somatic hybrid plants was demonstrated.

 

Keywords: tomato, Lycopersicon, L. esculentum, L. chilense, L. peruvianum, somatic hybrid, protoplast fusion

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 524-532

 

 

Agrobacterium tumefaciens-mediated Transformation

of Chickpea  (Cicer arietinum L.) using

Mature Embryonic Axes and Cotyledonary Nodes

 Indraneel Sanyal, A K Singh and D V Amla*

 

The mature embryo axes and cotyledonary nodes of chickpea (Cicer arietinum L.) were evaluated for Agrobacterium-mediated transformation and the production of stable transgenic plants expressing the reporter gene GUS was documented. The major limitation in delivering the T-DNA in these explants of grain legume has been the low frequency and inconsistency. With manipulation of co-cultivation conditions and preparation of excised explants with exposed regenerative cells in L2 and L3 layers followed by the stringent screening on selection pressure of 100-150 mg l-1, kanamycin exhibited significantly higher transformation frequency as indicated in the transient GUS expression. Amongst various strains of Agrobacteria, the strain, LBA 4404 was found to be most suitable for maximal transfer of T-DNA and minimum induction of hypersensitive response into the excised explants of chickpea. Kanamycin resistant chickpea shoots selected after 3-4 cycles of kanamycin screening were grafted onto 8-day-old seedlings. Mature plants recovered thereafter showed integration of T-DNA in PCR and Southern analysis and expression of GUS gene in histochemical assay. Screening of putative transformants of chickpea harbouring modified gfp gene (pCAMBIA 1303) at an early stage was possible on the basis of green fluorescence but the transformants failed to grow further. The quantitative evaluation of GUS activity in primary transformants showed expression levels ranging from 580-1950 p mol methyl umbelliferone (MU) per mg protein per min. Out of 22 primary transformants, only six showed setting of F1 seeds while others failed to produce mature seeds.

 

Keywords: chickpea, Agrobacterium tumefaciens, β-glucuronidase, transformants, Cicer arietinum L., southern hybridization

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 533-538

 

 

Expression of Capsid Precursor Polypeptide (P1) of Foot

and Mouth Disease Virus Vaccine Strains in Pichia pastoris

 R Renji, V Balamurugan, S N Saha, G R Reddy and V V S Suryanarayana*

 

Foot and mouth disease virus (FMDV) is the aetiological agent of a highly contagious vesicular disease of cloven-hooved animals. The gene coding for the capsid polyprotein (P1) of FMDV from vaccine strains was amplified and cloned into the Yeast transfer vector (pPIC-9K) at Eco RI and Not I. The cloned P1 gene was characterized by colony PCR, restriction enzyme digestion and sequence analysis. The recombinant plasmid of each serotype was transferred into GS115 strain of Pichia by electroporation. The His+ Pichia transformants were analysed for the presence of the insert in the yeast genome by PCR. PCR positive clones of each serotype were grown separately and expression was induced with 0.5% methanol. The expressed gene product in yeast, Pichia pastoris was characterized by SDS-PAGE and Western Blot analysis. This is the first report on the production of FMDV structural proteins in yeast.

 

Keywords: FMDV; serotypes; p1polyprotein; Pichia pastoris; expression

 

 

 

 

 Indian Journal of Biotechnology 

Vol 2(4), October, pp. 539-542

 

Comparison of Insecticide Resistant and

Susceptible Populations of Spodoptera litura Fab.

 S Janarthanan*, S Seshadri, K Kathiravan and S Ignacimuthu

 

Esterase activity of various populations of Spodoptera litura, collected from cotton fields of different regions of Tamil Nadu, was compared using a- and b-naphthyl acetate as substrates for enzyme reaction. Among the ten populations analyzed, five populations showed elevated activities for esterases. The RAPD-PCR analysis of resistant and susceptible populations revealed the existence of polymorphism. Specific amplification for esterase gene using custom made primers produced amplified product in resistant populations.

 

Keywords: Spodoptera litura , esterase, RAPD-PCR

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 543-548

 

Induced Immunity against Haemolymph Proteins of

Anopheles stephensi: Effects on Fecundity and

Transmission Blocking of Malaria Parasite

 Monika Gulia and S K Gakhar*

 

Rabbit antibodies to nine antigens (100, 88, 80, 64, 55, 43, 29, 23 and 15 kDa), derived from haemolymph of Anopheles stephensi mosquitoes after first gonotrophic cycle, tended to reduce the number of eggs produced by the mosquitoes and block the transmission of malaria parasite, Plasmodium vivax. Cross-reactivity of these antibodies was checked both by Western blotting and in vivo ELISA. In addition, effect of these antibodies on mortality, engorgement and hatchability in mosquitoes was also determined. The results indicated that haemolymph antibodies have the potential to disrupt the reproductive physiology of mosquitoes and further studies are needed with the target antigens.

 

Keywords:  Anopheles, haemolymph, antibodies, parasite, transmission-blocking

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 549-552

 

 

Expression of Recombinant Fatty Acid Binding
Protein from Fasciola gigantica  

D Sriveny, O K Raina*, Jaya Verma and S C Yadav

 

Fatty acid binding protein (FABP) is an attractive vaccine candidate and a drug target for controlling fasciolosis caused by Fasciola gigantica in ruminants. The cDNA encoding F. gigantica FABP was amplified by polymerase chain reaction and expressed in Escherichia coli as a recombinant protein using a prokaryotic expression system. The protein was purified to homogeneity by nickel chelating affinity chromatography and subsequently by ion-exchange chromatography. The recombinant protein thus obtained will be assessed for immuno-prophylaxis and for its immunodiagnostic potential in ruminants against F. gigantica.

 

Keywords: Fasciola gigantica, recombinant fatty acid binding protein, nickel chelating affinity chromatography, immuno-prophylaxis, immunodiagnosis

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 553-557

 
Protein Polymorphisms in Mastomys
[Praomys (mastomys) coucha]  

B Maity*, N Gupta and P Y Guru

 

Outbred mastomys [Praomys (mastomys) coucha], were examined at 15 chromosomal loci encoding proteins and enzymes using cellulose acetate electrophoresis (CAE) to establish protein polymorphisms and allelic patterns. Protein polymorphisms were found in five protein biochemical markers (Akp-1, Ce-2, Idh-1, Pgm-1 and Trf-1). As a result, inter species genetic variations in terms of observed heterozygosity (Hob=0.020±0.036) and expected heterozygosity (HE=0.136±0.205) were also observed. But polymorphic Idh-1 was in very low frequency (average af = 0.487). Alb-1, Akp-1, Ce-2, Car-1, Es-2, Hbb, Idh-1, Pgm-1, Pep-3 and Trf-1 had two alleles (a/b or ab), whereas Es-1, Gpi-1 and Mod-1 had single allele (a/b), and C-3, Es-3, Idh-1 had three alleles (abc). The genetic variations as well as allele frequencies within the strain may be useful for the biomedical research for proper designing of the study protocols. This study may also help the animal breeders to establish inbred lines of this strain in India on the basis of dominant/recessive alleles as per experimental requirements.

 

Keywords: protein polymorphisms, allelic pattern, cellulose acetate electrophoresis, mastomys

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 558-563

 

 

Nitrogen Stress Induces Magnaporthe grisea
(Hebert) Barr to Secrete Protoplast-Disrupting Proteins
R Rathour*, B M Singh and P Plaha

 

Experiments were conducted to determine the effect of nitrogen stress on the gene expression of Magnaporthe grisea (Hebert) Barr, a casual agent of blast disease of rice and other graminaceous species. Concentrated culture filtrate extracts of the fungus grown in nitrogen deficient medium exhibited strong disruptive activity against the protoplasts of different plant species including rice. However, the same activity was not observed in culture filtrate extracts of the fungus grown in nitrogen rich medium. The results suggest that nitrogen stress induces the fungus to secrete protoplast-disrupting factors. The nitrogen stress elicited protoplast-disrupting activity was found to be conserved among different host-limited forms of the pathogen. The microscopic events preceding the protoplast death and the rapidity of the response evinced plasma membrane to be the site of action of protoplast-disrupting factors. Preliminary characterization of culture filtrate extracts suggested the heat labile proteins of 20.4 to 22.4 kDa to be the prime candidates for protoplast-disrupting factors.

 

Keywords: Magnaporthe grisea, nitrogen stress, protoplast

 

 

 

 

 Indian Journal of Biotechnology 

Vol 2(4), October, pp. 564-570

 

 

Optimization of Medium and Cultural Conditions
for Neomycin  Production
using Response Surface Methodology

K Adinarayana*, P Ellaiah, B Srinivasulu, J Lakshmi Narayana and

K V V S N Bapi Raju

 

Optimization of medium ingredients and cultural conditions for maximum neomycin production was carried out using a new species, Streptomyces marinensis. The C source (maltose), the N source (sodium glutamate) and the cultural conditions such as pH, temperature and agitation (rpm) were selected for optimization. Full factorial composite experimental design and response surface methodologies were used in the design of experiments and in the analysis of results. The optimum values for the tested variables for maximum neomycin production were: maltose, 51.67 g l-1, sodium glutamate, 12.36 g l-1, pH, 7.48, temperature, 30.6°C and agitation of the shake flask 174 rpm. The maximum neomycin production was 7815 mg l-1. This method was efficient; only 36 experiments were necessary to assess these conditions, and model adequacy was very satisfactory, as the coefficient of determination was 0.9448.

 

Keywords: optimization; central composite design; response surface methodology; neomycin production

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 571-576

 

 

Studies on Hydantoinase Production using
Pseudomonas Strain
B G Gaikwad*, S Nene and B D Kulkarni

 

Studies were carried out using Pseudomonas sp. NCIM 2875 strain to achieve optimum growth and hydantoinase activity. A medium containing glucose-NaNO3 with supplement of salts, yeast extract (0.1%) and peptone was  developed. Maximum cell (wet wt) yield and hydantoinase activity was 12.7 g l-1 and 4.63 units (mM of N-carbamoylphenylglycine formed/min/g wet wt cells), respectively. The activity was higher than that obtained after cultivation of strain on nutrient broth medium.

 

Keywords: Pseudomonas sp., hydantoinase, medium optimization, semi-synthetic medium, D, L-phenylhydantoin, N-carbamoylphenylglycine

 

 

 

 

 Indian Journal of Biotechnology 

Vol 2(4), October, pp. 577-582

 

Use of Heavy Metals for Quantification of
Rhizobia and Suppression of Bacterial Contaminants
in Carrier Based Inoculants

R P Jeevan Kumar and K S Jauhri*

 

Quality analysis of commercial legume inoculants involves determination of number of live rhizobia of appropriate species per unit weight of carrier. Conventional analysis is performed by means of plate counts of rhizobia on CRYEMA, but contaminants sometimes suppress/mask the growth of rhizobial colonies and make their counts unrealistic. Plant infection-most probable number (MPN) assay is considered to be more reliable, but is relatively expensive and time consuming. In the present study, a selective medium is devised, which permits growth of selected strains of rhizobia and at the same time refrains the growth of contaminants on plates. To inhibit non-rhizobial contaminants in carrier based inoculants, heavy metals were added in different combinations. A combination of Zn and Co at a concentration of 50 ppm each was found to inhibit bacterial contaminants without impeding the growth of rhizobial colonies on CRYEMA. However, these additions were not effective in refraining the proliferation of contaminants in charcoal-soil based rhizobial inoculants at this concentration. These metals when used in higher concentrations in charcoal-soil based inoculants, adversely affected nodulation and the dry matter yield of soybean.

 

Keywords: heavy-metal, bacterial contaminant, inhibition, Rhizobium-inoculant

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 583-586

 

 

Isolation and Characterization of Aluminium and
Copper Resistant ‘P’ Solubilizing Alkalophilic Bacteria

 Shilpi Mittal, Jean-Marie Meyer and Reeta Goel*

 

Aluminium and copper resistant bacteria, isolated from contaminated soil, showed metal tolerance level of 3106 mM and 4398 mM, respectively as evident by four select isolates based on their phosphorus solubilization potential (0.2-6.4 mg mL-1). All four isolates could grow in a pH range of 5-11. Three isolates are member of Pseudomonadaceae family, Pseudomonas sp. (CD7), P. pseudomalli (CG13) and P. maltophila (TH18). The isolates, CD7 and CG13, belong to siderovar group as the Pseudomonas sp. strain B10, and are able to grow at 42°C. The CG13 is osmotolerant (10% NaCI). These isolates could be better bioinoculant candidate(s) for the contaminated alkalophilic sites.

 

Keywords: metal resistance, growth promotory microorganisms, phosphorus solubilization, siderotyping, osmotolerant, alkalophile

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 587-590

 

Evaluation of Multiplex Reverse Transcription
Polymerase Chain Reaction (RT-PCR) for Simultaneous Detection of Potato Viruses and Strains

Yogita Verma, Shivali Sood, Y S Ahlawat*, S M Paul Khurana, X Nie and R P Singh

 

A multiplex reverse transcription polymerase chain reaction (RT-PCR) protocol was evaluated for the simultaneous detection of Potato leaf roll virus (PLRV), Potato virus X (PVX) and Potato Virus Y (PVY). The multiplex RT-PCR detection of viruses was equally efficient whether RNAs were extracted using commercial kits or a sodium sulphite based nucleic acid extraction procedure. cDNAs were prepared either using a common primer (oligo dT) or specific antisense primer followed by specific primer pairs for PCR. The multiplex RT-PCR separation of strains of PVY was accomplished by using a competitive multiplex RT-PCR (with one antisense and two sense primers). The multivirus or multistrain detection approaches described here have potential application to other crop-virus combinations.

 

Keywords: detection of viruses, multiplex RT-PCR, potato viruses, strains

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 591-595

 

 

Plant Defence Activators Inducing Systemic Resistance in Zingiber officinale Rosc. against Pythium aphanidermatum (Edson) Fitz.

N C Karmakar, Rajyasri Ghosh and R P Purkayastha*

 

Selected plant defence activators were employed to induce systemic resistance in a susceptible cultivar of ginger against Pythium aphanidermatum (Edson) Fitz., a severe rhizome rot causing pathogen. Among the 6 cultivars of ginger tested, Varada was most susceptible, followed by Suprabha and Maran. Prior to sowing, soaking of rhizome seeds for 1 hr in 5 mM salicylic acid (SA), DL-b-aminobutyric acid (BABA) or 2,1,3-benzothiadiazole (BTH), significantly reduced the disease. Systemic protection against disease was evident even after 8 weeks of treatment. Analysis of leaf proteins of non-inoculated, inoculated and activator-treated both non-inoculated and inoculated plants (cv. Suprabha) by SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and Image Master VDS-ID gel analysis version 3.0 showed 18 protein bands including 3-5 defence related proteins having molecular masses 67, 56, 32, 20 and 14 kDa. These defence proteins increased in activator-treated inoculated plants and enhanced the resistance against P. aphanidermatum. In vitro growth response of P. aphanidermatum to different conc. of SA, BABA and BTH was tested. SA and BABA, respectively were the most and least inhibitory to the fungus.

 

Keywords: ginger, Pythium aphanidermatum, rhizome rot, defence activators, systemic resistance

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 596-602

 

 

 

Caspase-3 like Protein in Wheat-Tilletia indica Dual Culture System as Potential Biomarker for Host Resistance to Karnal Bunt

  Niroj Kumar Sethy, Dinesh Pandey, U S Singh and Anil Kumar*

 

Immuno-histochemical staining of dual co-cultured calli of wheat-Tilletia indica having resistant and susceptible line using anti-mycelial and anti-teliospore polyclonal antibodies revealed the differential resistance response as indicated by pathogen invasion, mycelial colonization, plasmolysis and immunolocalization of chlamydospore like structures. The p53 and caspases, crucial proteins of cell death pathways were detected by immunological procedures using heterologous immunoprobes in fungal colonized wheat calli. There was no difference in p53 expression in resistant and susceptible lines at different days after inoculation (DAI) of T. indica on calli raised from mature embryos. The expression of p53 started 14 DAI, increased steadily up to 21 DAI and became constant at 30 DAI showing the saturation of expression level. Studies on the expression of caspases revealed the absence of caspase-1 like protein at different stages of growth. The caspase-3 like protein was induced at 3 DAI in both hosts and its expression steadily increased at 14 DAI and reached a plateau at 21 DAI. A remarkable difference in the level of expression of this protein between resistant and susceptible co-cultured calli was observed. Differential activation of caspase-3 like protein was observed in 10 hosts and non-hosts. It was found to be correlated with the degree of susceptibility and the expression of resistance at cellular level. Hence, it could be used as a potential biomarker for identification of Karnal Bunt resistance sources.

 

Keywords: wheat, dual culture, sporidia, Karnal bunt, Tilletia indica, resistance, bio-marker, caspase-3, p53, immunoassay

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 602-606

 

 

Design and Optimization of Novel Immuno-dipstick Test
for Detection of Teliospores of Karnal Bunt
(Tilletia indica) of Wheat  

Ravi Kesari and Anil Kumar*

 

An immuno-dipstick test was developed for detection of teliospores of Karnal bunt using crude and DE-52 purified anti-teliospore antibodies. For development of this test, intact teliospores and solubilized teliosporic antigen were coated on nitrocellulose pad of dipsticks. On observation of the nitrocellulose pad under a stereo zoom microscope, the intact teliospores were found stuck on the pad after coating but got washed away with first washing. The positive tests after immunoprocessing of dipsticks indicate that surface antigen may tear off from teliospores and remains bound to nitrocellulose pad. Standardization and optimization of the test was done following antigen concentration kinetics and antibody dilution analysis as well as antibody incubation time. The test was also performed for checking cross-reactivity with other fungal pathogens. DE-52 purified antiserum gave partial reactivity with spores of only Tilletia barclayana and Puccinia recondita. The sensitivity of test was found to be 5 teliospores and 10 ng for solubilized teliosporic antigen.

 

Keywords: Karnal bunt, teliospore, polyclonal antibodies, immunodetection, immuno-dipstick test

 

 

 

 Indian Journal of Biotechnology 

Vol 2(4), October, pp. 607-609

 

Strain Differentiation of Salmonella Bareilly by AP-PCR  

M K Saxena, V P Singh, Anjani Saxena, B D Lakhchura and B Sharma*

 

Several PCR based methods such as AP-PCR, ERIC, ERIC-PCR, REP-PCR and RAPD have been used for bacterial strain differentiation and identification. These methods are rapid, reliable and economical. In the present study, out of 10 strains of Salmonella Bareilly differentiated by AP-PCR, seven profiles were obtained. The study indicates that AP-PCR is an efficient tool for strain differentiation and can be used for molecular epidemiological studies. This is the first report in India in which AP-PCR has been used for molecular typing of Salmonella Bareilly.

 

Keywords: molecular typing, ribotyping, AP-PCR, ERIC-PCR, REP-PCR

 

 

 

 

Indian Journal of Biotechnology 

Vol 2(4), October, pp. 610-612

 

Biotransformation of Ferulic Acid to Vanillin by Locally Isolated Bacterial Cultures  

Renu Agrawal, Y N Seetharam, R Chandrakanth Kelamani* & G Jyothishwaran

 

The biotransformation of ferulic acid to vanillin was carried out using locally isolated bacterial cultures from manure, paddy straw and garbage samples which include Pseudomonas sp. Pseudomonas sp. was enriched with the ferulic acid thrice and subsequently named as EF1, EF2 and EF3. EF3 produced maximum vanillin (850 µg/100mg culture), followed by EF2 (842 µg/100mg culture), manure isolate MAMWR (B) (598 µg/100mg culture), EF1 (474 µg/100mg culture) and Pseudomonas sp. (230 µg/100mg culture). Garbage isolate GRMWSR (B) produced very insignificant amount (1.13 µg/100mg culture) of vanillin.

 

Keywords: biotransformation, ferulic acid, Pseudomonas sp., vanillin

 

 

 

 

 Indian Journal of Biotechnology 

Vol 2(4), October, pp. 613-615

 

 

High-rate Vermicomposting Systems for
Recycling Paper Waste  

S Gajalakshmi and S A Abbasi*

 

Composted paper waste was vermicomposted with Eudrilus eugeniae Kinberg in reactors with higher earthworm densities (62.5-162.5 animals l-1) than used in conventional vermireactors (7 animals l-1). Continuous operation of such ‘high-rate’ vermireactors for significantly long periods (7 months) resulted in consistently high vermicast output (72-81%) of the feed being turned to vermicasts in reactors of different earthworm densities with very little animal mortality (<4.4%). The earthworms consistently gained weight and produced offsprings in all the reactors, throughout the experiment. These findings establish the sustainability of high-rate vermireactors fed on paper waste.

 

Keywords: compost, paper waste, vermicompost, earthworm, high-rate

 

 

 

 

 

AUTHOR INDEX

 

Abbasi, S A

613

Janarthanan S

539

Purkayastha R P

591

Abdin, M Z

477

Jauhri K S

577

 

 

Adinarayana K

564

Jeevan Kumar R P

577

Raina O K

549

Agrawal R

610

Jyothishwaran G

610

Rathour R

558

Ahlawat Y S

587

 

 

Reddy G R

533

Amla D V

524

Karmakar N C

591

Renji R

533

Ashok C

499

Kathiravan K

539

 

 

 

 

Kelamani R C

610

Saha S N

533

Balamurugan V

533

Kesri R

603

Sanyal I

524

Bapi Raju K V V S N

564

Kulkarni B D

571

Saxena A

607

Bhaskara Rao Y U

494

Kumar A

596, 603

Saxena M K

607

 

 

 

 

Seetharam Y N

610

Cocking E C

504, 512

Lakhchura B D

607

Seshadri S

539

 

 

Lakshmi Narayana J

564

Sethy N K

596

Davey M R

504, 512

 

 

Sharma B

607

 

 

Maity B

553

Singh A K

524

Ellaiah P

564

Meyer J-M

583

Singh B M

558

 

 

Mittal S

583

Singh R P

587

Gaikwad B G

571

 

 

Singh U S

596

Gajalakshmi S

613

Nasser M W

477

Singh V P

607

Gakhar S K

543

Nene S

571

Sood S

587

Geetha M S

499

Nie X

587

Srinivasulu, B

564

Ghosh R

591

 

 

Sriveny D

549

Goel R

583

Pandey D

596

Sullia S B

499

Gulia M

543

Patil R S

504, 512

Suryanarayana V V S

533

Gupta N

553

Paul Khurana S M

587

 

 

Guru P Y

553

Plaha P

558

Verma J

549

 

 

Pooja V

477

Verma Y

587

Ignacimuthu S

539

 

 

 

 

 

 

Power J B

504, 512

Yadav S C

549

Jain S K

477