Indian Journal of Biotechnology

 

 

VOLUME 1

NUMBER 4

OCTOBER 2002

 

CONTENTS

 

Review

 

Trends in immobilized enzyme and cell technology

321

        S F D’Souza

 

 

 

Papers

 

Effective protocol for Agrobacterium–mediated leaf disc transformation in tomato (Lycopersicon esculentum Mill.)

339

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

 

 

 

Factors influencing GUS expression in cucumber (Cucumis sativus Linn.)

344

        A Vasudevan, A Ganapathi, N Selvaraj & G Vengadesan

 

 

 

Sequence conservation in the coat protein gene of Tobbacco streak virus isolates causing necrosis disease in cotton, mung bean, sunflower and sunnhemp in India

350

        A I Bhat, R K Jain, V Chaudhary, M Krishna Reddy, M Ramiah, S N Chattannavar & A Varma

 

 

 

Morphological changes in submerged cultivation of Aspergillus awamori

357

        for glucoamylase production

 

        David K Daniel, Nabanita Biswas  & Debabrata Das

 

 

 

Dac-Elisa technique for early detection of red rot pathogen in sugarcane
var. CoC 671

363

        Lingayya Hiremath & G R Naik

 

 

 

Influence of ammonium nitrate on plant regeneration in Indica rice (Oryza sativa Linn.)

367

        Ashok Kumar Sahrawat & Suresh Chand

 

 

 

Piriformospora indica—in vitro raised leguminous plants: A new dimension in establishment and phyto-promotion

372

        Archana Singh, Anjana Singh & Ajit Varma

 

 

 

PCR based re-amplification step for detection and characterization of fluorescent Pseudomonads by ARDRA

377

        M Kumar, S K Goel & Reeta Goel

 

 

 

Rosmarinic acid synthesis in shoot cultures of Mentha arvensis Linn.

 

        Savita V Phatak & Mohan R Heble

381

 

 

 

Diagnostic tests on the mode of ligand binding to proteins: Application to
Zymomonas mobilis strains          

 

        E M Papamichael, A-I Koukkou, E Douka, G Vartholomatos, K A Masavetas &
C Drainas

 

 

 

Comparative study on transformation of azo dyes by different white rot fungi

393

        Pradeep Verma & Datta Madamwar

 

 

 

Short Communications

 

Differentiation of Indian isolates of Equine Herpes Virus (EHV-1) by restriction endonuclease digestion

397

        Birendra K Singh, B R Gulati & B Poonia

 

 

 

In vitro shoot regeneration from leaf and nodal explants of Enicostemma hyssopifolium (Willd.) Verd.—A vulnerable medicinal plant

401

        Y N Seetharam, Aravind Barad, Gururaj Chalegeri, G Jyothishwaran,
Kiran S Ghanti & Venugopal Bhakri

 

 

 

News Scan

 

Future fuel of India: Bio-ethanol

405

        P D Tyagi

 

 

 

Conference Report

408

Announcement

410

Annual Index

411

Instructions to Contributors

422

 

 

 


 


Trends in Immobilized Enzyme and Cell Technology

S F D’Souza*

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

 

Enzyme and microbial technology has influenced the process industry significantly in the recent years by improvement of existing processes as well as in the development of new eco-friendly industrial bioprocesses. One of the techniques, which have played a significant role, is the immobilization of enzymes and cells. Immobilization helps in the retention of the biomass in a reactor geometry thus enabling in their economic reuse and in the development of continuous processes. Immobilization also improves stability and prevents product contamination thus paving the use of crude enzyme preparations like whole cells in bioprocessing. Protection of cells from environmental perturbations on immobilization has helped to introduce them into soils for agricultural and environmental applications. In the fabrication of biosensors immobilization helps in establishing intimate contact of the biomaterial on transducer surface and in medicine for the formation of immunobarrier. The current review delineates some of these aspects.

Keywords: bioprocessing, bioremediation, biosensors, enzyme stabilization, fermentation, immobilized cells, immobilized enzymes, immobilization techniques

 

Effective Protocol for Agrobacterium–mediated Leaf Disc Transformation in Tomato (Lycopersicon esculentum Mill.)y

 

R S Patil1*, M R Davey2, J B Power2 and E C Cocking2

1Department of Horticulture, Mahatma Phule Krishi Vidyapeeth, Rahuri 413 722, India

2Plant Genetic Manipulation Group, Life Science Department, University of Nottingham, NG7 2RD, Nottingham, UK

An efficient transformation system for Indian tomato cultivar, ‘Pusa Ruby’ was developed by identifying and optimizing various parameters affecting transformation rates, non-transformed growth and bacterial over growth. A protocol consisting leaf explants from 14-day-old in vitro seedlings, 2 day pre-conditioning on culture medium, 2 day co-cultivation period for inoculated explants, 100 μm l-1 Kanamycin Sulphate and culture medium containing MS salt with 0.1 mg l-1 IAA, 1.0 mg l-1 Zeatin, 2% (w/v) sucrose, pH 5.8 resulted in significant higher transformation rate (66.7%) within 4-6 weeks period. Confirmation of transformation was carried out in protoplast, cell, tissue and shoot cultures by Kanamycin resistance; nopaline assay, NPT-II assay and PCR analysis with primers specific to npt-II gene. This system will be certainly exploitable for transferring agronomically useful genes in tomato cultivars.

Keywords: Agrobacterium, genetic transformation, tomato, Lycopersicon esculentum

 

Factors Influencing GUS Expression in Cucumber (Cucumis sativus Linn.)

 

A Vasudevan1, A Ganapathi1*, N Selvaraj2 and G Vengadesan1

1Department of Biotechnology, Bharathidasan University, Tiruchirappalli 620 024, India

2Department of Botany, Periyar E V R College (Autonomous), Tiruchirappalli 620 023, India

 

The transformation efficiency of two strains of Agrobacterium tumefaciens, EHA 105 and LBA 4404 with three cucumber cultivars, viz. Endeavor, Green Long and Poinsett 76 have been investigated. Factors such as age of explants, co-cultivation period, efficiency of strains, acetosyringone treatment, use of Kanamycin and Basta as selection agents were assessed. GUS histochemical assay was performed to assess transformation efficiency. 5-day-old cotyledons and 3-day co-cultivation period for Endeavor and Green Long and 2-day co-cultivation period for Poinsett 76 were suitable for efficient transformation. EHA 105 was more virulent and the transformation efficiency was two to three folds higher than LBA 4404. Among the three cultivars, Endeavor was found to be more responsive to transformation as evidenced by GUS expression followed by Green Long and Poinsett 76. Acetosyringone (20 mM- 50 mM) enhanced the efficiency of transformation in all the three cultivars. Basta at 10 mg/l was more efficient selection agent than Kanamycin at 100 mg/l. Shoot regeneration occurred within 6 weeks after co-cultivation.

Keywords: Cucumis sativus, Agrobacterium, acetosyringone, Kanamycin, Basta

 

Sequence Conservation in the Coat Protein Gene of Tobacco streak virus Isolates Causing Necrosis Disease in Cotton, Mung bean, Sunflower and Sunnhemp in India

A I Bhat1, R K Jain5*, V Chaudhary5, M Krishna Reddy2, M Ramiah3, S N Chattannavar4 and A Varma5

1Indian Institute of Spices Research, Marikunnu, Calicut 673 012, India

2Indian Institute of Horticultural Research, Bangalore 560 089, India

3Tamil Nadu Agricultural University, Coimbatore 641 003, India

4University of Agricultural Sciences, Agricultural Research Station, Dharwad 580 005, India

5Advanced Center for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110 012, India

 

Natural infection of Tobacco streak virus (TSV) in cotton, mung bean, sunflower and sunn-hemp, collected from different locations in India, was detected by reverse transcription-polymerase chain reaction (RT-PCR). The coat protein (CP) gene sequences of the six TSV isolates originating from different hosts and locations were amplified. The resulting amplicons were cloned and sequenced to assess molecular variability. The sequence and phylogenetic analyses revealed that the CP gene among TSV isolates collected from different hosts and locations was highly conserved (99-100%), suggesting a common origin.

             Keywords: Tobacco streak virus, cotton, mung bean, sunflower, sunn-hemp, coat protein gene sequence

 

 

Morphological Changes in Submerged Cultivation of Aspergillus awamori for Glucoamylase Production

David K Daniel1, Nabanita Biswas2 and Debabrata Das3*

1Department of Chemical Engineering, Vellore Institute of Technology, Vellore 632 014, India

2Department of Microbiology, Calcutta University, Kolkata 700 019, India

3Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India

 

Industrial processes involving submerged fermentation of fungal cultures require greater attention due to complex morphologies. Growth of Aspergillus awamori was investigated in submerged culture using different parameters such as pH and agitator speed with a goal to assess the morphology and fragmentation behaviour during the fermentation. Microscopic examination of the mycelia revealed that the length of the branches decreased with increasing pH. Pellet formation was significant at pH 5.5 and the diameter of the pellet was found to vary with fermentation time. A maximum pellet diameter of 1.69 mm was observed at a pH of 7.5, which decreased to 0.38 mm for cultivations at pH of 3.5. During mechanical agitation, fragmentation was found to dominate fungal growth and branching. Agitator speeds higher than 600 rpm resulted in decreased cell growth and glucoamylase production.

 

Keywords: Aspergillus awamori, fungal morphology, glucoamylase, fragmentation, pellets, agitation

 

Dac–Elisa Technique for Early Detection of Red Rot Pathogen in Sugarcane var. CoC 671

Lingayya Hiremath and G R Naik*

Department of Biotechnology, Gulbarga University, Gulbarga 585 106, India

The sugarcane variety CoC 671 is an early maturing, high sugar content variety in India. Recently, it has become very susceptible to red rot caused by Colletotrichum falcatum Went. To detect the pathogen at an early stage, a new serological technique, enzyme–linked immunosorbent assay was developed based on antiserum developed against the protein of host pathogen. Direct antigen coating enzyme–linked immunosorbent assay (Dac–Elisa) for the early detection of red rot infection has been standardized. Further, this technique was found reliable to screened planting material of sugarcane variety CoC 671 for red rot infection at an earlier stage. Besides, in vitro red rot treated as well as healthy calli were also screened for red rot infection.

Keywords: Colletotrichum falcatum, Dac-Elisa, red rot pathogen, sugarcane

 

 

Influence of Ammonium Nitrate on Plant Regeneration in Indica Rice (Oryza sativa Linn.)

 

Ashok Kumar Sahrawat and Suresh Chand*

Plant Tissue Culture & Genetics Research Group, School of Life Sciences, Devi Ahilya University,
Khandwa Road Campus, Indore 452 017, India

The effect of ammonium nitrate (an essential macrosalt) on plant regeneration from mesocotyl segments of indica rice (Oryza sativa Linn. cv. Safari-17) has been investigated. Callus developed from the cut ends of mesocotyl segments on MS medium supplemented with 2,4-D (20 mM), BAP (2.2 mM) and various concentrations of ammonium nitrate (10-80 mM). Highly organised and nodular callus developed after transfer of morphogenic calli to the MS medium containing 5 mM 2,4-D, 2.2 mM BAP and ammonium nitrate (20-80 mM). The presence of ammonium nitrate was found to be essential for the induction and proliferation of embryogenic callus. Shoot differentiation occurred after transfer of calli to the MS medium containing 10, 20 (MS original), 40 and 80 mM ammonium nitrate alone or in combination with 8.8 mM BAP. No shoot regeneration occurred in the absence of ammonium nitrate. The highest frequency of shoot regeneration and average number of shoots per callus clump was obtained on MS medium containing 40 mM ammonium nitrate. No significant difference in regeneration frequency and average number of plantlets per callus clump was observed when BAP (8.8 mM) was incorporated in the regeneration medium containing various levels of ammonium nitrate. Regenerated shoots were rooted on MS medium containing 4.9 mM IBA. Rooted plantlets were established to soil. The results show that ammonium nitrate play an important role in plant regeneration in indica rice.

Keywords: Oryza sativa, mesocotyl segments, ammonium nitrate, morphogenic callus, plant regeneration

 

 

Piriformospora indica in vitro Raised Leguminous Plants: A New Dimension in Establishment and Phyto-promotion

 

Archana Singh1, Anjana Singh2 and Ajit Varma2*

1Department of Biological Sciences, Michigan Technological University, 1400 Townsend Drive, Houghton, Michigan 49931, USA

2School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

 

Piriformospora indica, a newly discovered root colonizing, AMF-like fungus, showed prominent positive influence on the economically important legumes, chickpea (Cicer arietinum Linn.) and green gram (Vigna radiata Linn.). Micro propagated wild and transgenic legume plants often fail to produce functionally developed root system. It is postulated that the interaction of this fungus, may promote the formation of physiologically active root system. The fungus can be mass multiplied axenically on a simplified synthetic medium for wide scale application with crops of economic importance.

Keywords: Piriformospora indica, root colonizing fungus, legumes, plant promotion, axenic culture, biological hardening

 

PCR based Re-amplification Step for Detection and Characterization of Fluorescent Pseudomonads by ARDRA

 

M Kumar1, S K Goel2 and Reeta Goel1*

1Department of Microbiology, G B Pant University of Agriculture & Technology, Pantnagar 263 145, India

2Industrial Toxicological Research Centre, Lucknow 226 001, India

 

The objective of the present study is to develop a rapid and precise method for monitoring and characterization of bacterial strain(s) at taxonomic level, using universal primers specific for 16S rRNA gene in a PCR based protocol. Authors have introduced an extra re-amplification step of PCR to study wild type fluorescent psuedomonads strains and their cold resistant mutants. Using normal PCR amplification when genomic DNA was amplified only a short fragment of 500 bp was obtained. However, when a re-amplification step (20 cycles) was done using the small volume (2 ml) of first cycle aliquot, authors observed two bands of ~1500 and 500 bp. The same was utilized for restriction digestion using restriction enzymes, Alu I, Pst I, Xba I, Hae III, Hind III, Bgl II and EcoRI separately. The restriction pattern indicating amplified ribosomal DNA restriction analysis (ARDRA) and re-amplification step used by authors seems to be a precise-technique for characterization.

Keywords: ARDRA, rDNA, PCR, CRM, CRPF

 

Rosmarinic Acid Synthesis in Shoot Cultures of Mentha arvensis Linn.

 

Savita V Phatak* and Mohan R Heble

The Kelkar Education Trust’s Scientific Research Centre, Mithagar Road, Mulund (E), Mumbai 400081, India

Rosmarinic acid synthesized by multiple shoot cultures of Mentha arvensis was evaluated in four different basal media, at various sucrose concentrations, at altered KH2PO4 levels, and in the presence of agents like phenylalanine or peptone. Shoots grown in liquid MS medium supplemented with 3% sucrose and phenylalanine (30 mg1-1) produced highest amount of rosmarinic acid (0.21 mg g -1) on fresh weight basis. This is the first report of synthesis of rosmarinic acid in the cultured shoots of Mentha arvensis.

Keywords: Japanese mint, Mentha arvensis, multiple shoot culture, rosmarinic acid

 

 

Diagnostic Tests on the Mode of Ligand Binding to Proteins: Application to Zymomonas mobilis Strains

 

E M Papamichael1*, A-I Koukkou1, E Douka1, G Vartholomatos1, K A Masavetas2 and C Drainas1

1 Department of Chemistry, University of Ioannina, Ioannina 451 10, Greece

2 Department of Chemical Engineering, Section of Materials Science and Engineering, Laboratory of Physical Chemistry, Iroon Polytechniou, National Technical University, 9 Zografou, Athens 157 80, Greece

The occurrence of biphasic responses when kinetic data, which describe the binding of ligands to proteins are outlined in Eadie - Hofstee plots are not uncommon as in several cases of glucose uptake of Zymomonas mobilis strain ATCC 10988 and its derivative CU1Rif2/clone. In this work the authors report two novel diagnostic tests, which can be used easily and routinely in distinguishing the two possible cases. In the first case, two proteinaceous components are involved having one binding site per protein molecule and in the second case a single proteinaceous component having two binding sites per protein molecule is involved. These tests are based on the evaluation of either the fractal dimension and/or the coefficients of a virial expansion, of the Michaelis-Menten equation.

Keywords: Zymomonas mobilis, diagnostic, fractal, virial,glucose uptake

 

Comparative Study on Transformation of Azo Dyes by Different White Rot Fungi

Pradeep Verma and Datta Madamwar*

Department of Biosciences, Sardar Patel University, vallabh Vidyanagar 388 120, India

 

A comparative study on the extracellular ligninolytic enzyme production and decolourization of Ranocid fast blue and Acid black 210 by three strains of white rot fungi, Phanerochaete chrysosporium, Trametes versicolor and Pleurotus ostreatus carried out in Bushnell and Hass medium under static conditions revealed differential production of ligninolytic enzymes and decolourization efficiency. T. versicolor and P. ostreatus showed laccase and manganese peroxidase (MnP) activity in culture supernatant while no lignin peroxidase (LiP) was detected. P. chrysosporium showed only LiP and MnP activities. No laccase activity was detected in culture supernatant. Transformation of Ranocid fast blue and Acid black 210 was determined by monitoring the decrease in absorbance at λmax of each dye. During transformation of azo dyes by different white rot fungi, metabolite formation was also monitored.

Keywords: white rot fungi, decolourization, laccase, manganese peroxidase (MnP), lignin peroxidase (LiP)

 

 

Differentiation of Indian Isolates of Equine Herpes Virus (EHV-1) by Restriction Endonuclease Digestion

 

Birendra K Singh1*, B R Gulati2 and B Poonia1

1National Research Centre on Equines, Sirsa Road, Hisar 125 001, India

2Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar 125 004, India

 

The genome of five local isolates of EHV-1 (Hisar-90-7, Jind-96, Tohana-96-2, Delhi-98 and Raj-98) and one reference strain 592 was digested with different restriction endonucleases (RE). On electrophoresis of digested DNA in 0.8% agarose gel, Raj-98 could be differentiated from other viral isolates using BamHI and KpnI. Reference strain (592) differed from other viruses in RE profile of HindIII (one band of ~3500 bp lacking) and XbaI (2 bands of ~4800 and ~2900 bp missing). This study indicates that at least two genetically variant isolates of EHV-1 are circulating amongst the equines of northern India.

Keywords: EHV-1, RE analysis, restriction endonucleases, genetic variation

 

 

In vitro Shoot Regeneration from Leaf and Nodal Explants of Enicostemma hyssopifolium (Willd.) Verd.¾A Vulnerable Medicinal Plant

Y N Seetharam, Aravind Barad*, Gururaj Chalegeri,
G Jyothishwaran, Kiran S Ghanti and Venugopal Bhakri

Biosystematics and Medicinal Plants Laboratory, Dept of P G Studies and Research in Botany, Gulbarga University,
Gulbarga 585 106, India.

Enicostemma hyssopifolium (Willd.) Verd. plants were established in aseptic cultures from surface sterilized leaf and single node stem segments. Multiple shoots were elicited from leaf explants on MS medium supplemented with BAP (1.5 mg 1-1) and IAA (0.5 mg 1-1), while from nodal explants on BAP (1.0 mg 1-1) and IAA (0.5 mg1-1). Maximum shoot proliferation and elongation was established in shoots derived from leaf explant on MS medium supplemented with KN
(1.0 mg l-l) and BAP (1.0 mg l-1), while in shoots
derived from nodal explant it was on MS supplemented with KN (1.0 mg 1-1) and BAP (0.5 mg 1-1). Plantlets were rooted on ½ strength MS medium supplemented with IAA (1.0 mg 1-1). The in vitro raised plantlets were acclimatized in green-house and successfully transplanted to the nursery.

 

Keywords: E. hyssopifolium, vulnerable, multiple shoot, proliferation, acclimatization