Indian Journal of Biotechnology

 

http://www.niscair.res.in

 

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VOLUME 6

CODEN:IJBNAR 6(4) 427-586  (2007)

NUMBER 4

OCTOBER 2007

ISSN:0972-5849

 

CONTENTS

 

Reviews

 

Human granulocyte-macrophage colony-stimulating factor: The protein and its current & emerging applications

 

435

IPC Code: Int. Cl.8 C12N15/09, 15/10, 15/11

 

Prasanta K Ghosh, Devesh Bhardwaj & Rucha Karnik

 

 

 

Pharmacogenomics: Translating functional genomics to personalized medicine

449

        IPC Code: Int. Cl.8 C12N15/00, 15/10

 

        Shalini Rajkumar

 

 

 

Papers

 

Genomic analysis of SNPs in breast cancer by using bioinformatics databases

456

            IPC Code: Cl.8 C12N15/00

 

            K Siva Sankar, N Satish Chandra Reddy, Kaiser Jamil & Vamsy C Mohana

 

 

 

Preparation and in vitro cytotoxic evaluation of taxol immunoconjugates

463

IPC Code: Int. Cl.8 A01N63/00, C12N15/00

 

K Prabhakar, S Ganesh, V Baheti, S Saisivam, Y Maadhusudan Rao & V Kishan

 

 

 

Statistical evaluation of medium components by Plackett-Burman experimental design and kinetic modeling of lipase production by Pseudomonas fluorescens

 

469

IPC Code: Int. Cl.8 C12N9/20

 

            Aravindan Rajendran, Meikandhan Thirugnanam & Viruthagiri Thangavelu

 

 

 

Covalent immobilization of lipase onto onion membrane affixed on plastic surface:         Kinetic properties and application in milk fat hydrolysis

 

479

IPC Code: Int. Cl.8 C12N9/20, 11/08

 

Vikas, R Kumar & C S Pundir

     

       

 

Homology modeling of putative thioredoxin from Helicobacetr pylori

485

IPC Code: Int. Cl.8 G01N33/00

 

D Premalatha, P Ravindra &L Venkateshwar Rao

 

 

 

Charcterization and screening of beneficial bacteria obtained on King’s B agar from tea rhizosphere

 

490

IPC Code: Int. Cl.8 C12N15/10

 

Tanushree Mazumdar, C Goswami & N C Talukdar

 

 

 

Isolation of pigeon pea (Cajanus cajan L.) legumin gene promoter and identification of conserved regulatory elements using tools of bioinformatics

 

495

IPC Code: Int. Cl.8 C12N15/09, 15/29

 

Rajani Jaiswal, Vikrant Nain, M Z Abdin & P A Kumar

 

 

 

Biolistic-mediated DNA delivery and transient expression of GUS in hypocotyls of Feronia limonia L.—A fruit tree

 

504

IPC Code: Int. Cl.8 A01H15/00; C12N15/09, 15/11

 

S D Purohit, S Raghuvanshi & A K Tyagi

 

 

 

Effect of salinity on growth and proteomic changes in two cultivars of mulberry (Morus alba L.) with contrasting salt tolerance

 

508

IPC Code: Int. Cl.8 8 G01N33/00

 

G Jyothsna Kumari, S Giridara Kumar, M Thippeswamy, A Annapurnadevi S Thimma Naik  & Chinta Sudhakar

 

 

 

Assessment of genetic diversity in Azadirachta indica based on DNA fingerprinting

519

IPC Code: Int. Cl.8 C12N15/10, 15/11

 

R S Dhillon, T Mohapatra, S Singh, K S Boora & K Singh

 

 

 

Optimization of guggulsterone production in callus cultures of Commiphora wightii (Arnott.) Bhandari

 

525

IPC Code: Int. Cl.8 A01H4/00; A61P3/06

 

Meera Mathur, A K Jain, S Dass & K G Ramawat

 

 

 

Plant regeneration in different genotypes of indica rice

532

IPC Code: Int. Cl.8 A01H4/00

 

Anusrita Biswas & Asit B Mandal

 

 

 

In vitro propagation of an endemic umbellifer, Hydrocotyle conferta

541

IPC Code: Int. Cl.8 A01H4/00

 

S Karuppusamy, V Aruna, C Kiranmai & T Pullaiah

 

 

         

An alternative source for regenerable organogenic callus induction in Jatropha curcas L.

545

IPC Code: Int. Cl.8 A01H4/00

 

Shilpa Rajore & Amla Batra

 

 

 

Protocol optimization for RAPD in cyanobacteria

549

        IPC Code: Int. Cl.8 C12N15/10

 

        Shalini, Saurabh Tiwari, Rajinder K Gupta, Sunil Pabbi & Dolly Wattal Dhar

 

 

 

Short Communications

 

 

 

Enzymatic characteristics of lignin peroxidases of indigenous lignolytic fungal strains—Part I

 

553

IPC Code: Int. Cl.8 A01G1/04; A01N63/04

 

V K Patel, R S S Yadav & K D S Yadav

 

 

 

Sericulture pupal waste—A new production medium for mass cultivation of Bacillus thuringiensis

 

557

IPC Code: Int. Cl.8 C12N1/16

 

A Karthikeyan & N Sivakumar

 

Molecular identification of Proutisia moesta as the vector and the phylogenetic analysis of KWD phytoplasma

 

560

IPC Code: Int. Cl.8 C12N15/09, 15/29

 

        Boby T Edwin & C Mohankumar

 

 

 

Thidiazuron induced shoot regeneration in the endangered species, Exacum travancoricum Beedi

564

IPC Code: Int. Cl.8 A01H4/00

 

P Kannan, A Premkumar & S Ignacimuthu

 

 

 

Comparative studies on the digestive enzymes in the gut of earthworms, Eudrilus eugeniae and Eisenia fetida

567

IPC Code: Int. Cl.8 C05F3/00, 9/04; C12N9/20

 

M Lakshmi Prabha,  Indira A Jayaraaj, R Jeyaraaj & Srinivasa Rao

 

 

 

List of Referees

571

 

 

Annual Author Index

577

 

 

Annual Subject Index

579

 

 

Annual IPC Code Index

582

 

 

Instructions to Contributors

583


 

 

AUTHOR INDEX

 


Abdin M Z

495

Annapurnadevi A

508

Aravindan R

469

Aruna V

541

 

 

Baheti V

463

Batra A

545

Bhardwaj D

435

Biswas A

532

Boora K S

519

 

 

Dass S

525

Dhar D W

549

Dhillon R S

519

 

 

Edwin B T

560

 

 

Ganesh S

463

Ghosh P K

435

Goswami C

490

Gupta R K

549

 

 

Ignacimuthu S

564

 

 

Jaiswal R

495

Jamil K

456

Jain A K

525

Jayaraaj I A

567

Jeyaraaj R

567

 

Kannan P

564

Karnik R

435

Karthikeyan A

557

Karuppusamy S

541

Kiranmai C

541

Kishan V

463

Kumar P A

495

Kumar R

479

Kumar G S

508

Lakshmi Prabha M

567

 

 

Mandal A B

532

Mathur M

525

Mazumdar T

490

Meikandhan T

469

Mohana V C

456

Mohankumar C

560

Mohapatra T

519

 

 

Naik  S T

508

Nain V

495

 

 

Pabbi S

549

Patel V K

553

Prabhakar K

463

Premalatha D

485

Premkumar A

564

Pullaiah T

541

Purohit S D

504

 

Raghuvanshi S

504

 

Rajkumar S

449

 

Rajore S

545

 

Ramawat K G

525

 

Rao M Y

463

 

Rao S

567

 

Rao V L

485

 

Ravindra P

485

 

Reddy N S C

456

 

 

Saisivam S

463

Shalini

549

Singh K

519

Singh S

519

Sivakumar N

557

Siva Sankar K

456

Sudhakar C

508

 

 

Talukdar N C

490

Thippeswamy M

508

Tiwari S

549

Tyagi A K

504

 

 

Vikas

479

Viruthagiri T

469

 

 

Yadav K D S

553

Yadav R S S

553


 

Reviews

Indian Journal of Biotechnology

Vol 6, October 2007, pp 435-448

 

 

Human granulocyte-macrophage colony-stimulating factor: The protein and its current & emerging applications

 

Prasanta K Ghosh*, Devesh Bhardwaj and Rucha Karnik

Department of Biotechnology, Cadila Pharmaceuticals Limited, 342, Nani Kadi, Mehsana 382 715, India

Received 10 August 2006; revised 2 March 2007; accepted 5 May 2007

Blood cells under a variety of conditions produce cytokines which regulate their proliferation, communication and functioning. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor mainly responsible for the proliferation of granulocyte-s and macrophages. It is a molecule of immense therapeutic interest due to its wide-ranging actions on immune cells. Recombinant human GM-CSF has been expressed, purified and characterized. It has diverse biological manifestations, which has led to its being explored for use in cancer therapy, AIDS therapy, as a vaccine adjuvant, in certain types of wound healing, promotion of collateral growth of coronary artery, usefulness in treating Crohn’s disease symptoms and in the management of a host of viral, bacterial and fungal infections. Newer therapeutic applications are also being unveiled. The physico-chemical as well as the biological features and the applications (current and emerging) of GM-CSF are discussed.

Keywords: GM-CSF, cytokines, T-cell induction, cytokines applications, GM-CSF applications, Crohn’s disease

IPC Code: Int. Cl.8 C12N15/09, 15/10, 15/11

Indian Journal of Biotechnology

Vol 6, October 2007, pp 449-455

 

 

Pharmacogenomics: Translating functional genomics to personalized medicine

Shalini Rajkumar*

Department of Biotechnology, Institute of Science,
Nirma University of Science and Technology Ahmedabad 382 481, India

Received 9 May 2006; revised 29 January 2007; accepted 28 March 2007

The emergence of human genome project brings hope of personalized medicine as patients with identical clinical symptoms may respond differently to the same drug therapy. Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors and other drug targets have been linked to differences in the efficacy and toxicity of many drugs between two individuals. Pharmacogenomics is the study of genetic factors that mediate a person’s drug response. Pharmacogenomic analysis promises to identify disease susceptibility genes thus discovering new drug targets. This may lead to an individualized application for drug therapy and bring new insights into disease prevention. The emerging discipline of pharmacogenomics attempts to apply the innovative technologies of genome sequencing in order to better understand drug response to produce more effective drugs with fewer side effects on the basis of individual patient’s genetic make up and making personalized medicine an economically viable possibility.

Keywords: pharmacogenomics, gene polymorphism, SNP, DNA sequencing, functional genomics

IPC Code: Int. Cl.8 C12N15/00, 15/10

Papers

Indian Journal of Biotechnology

Vol 6 October 2007, pp 456-462

 

 

Genomic analysis of SNPs in breast cancer by using bioinformatics databases

K Siva Sankar1, N Satish Chandra Reddy1, Kaiser Jamil1,2* and Vamsy C Mohana2

1Genetics Department, Bhagwan Mahavir Medical and Research Centre, Hyderabad 500 004, India

2Research Wing, Indo American Cancer Institute and Research Centre, Hyderabad 500 034, India

Received 20 December 2005; revised 27 November 2006; accepted 5 February 2007

The role of single nucleotide polymorphs (SNPs) in genes that modulate or promote cancer process has not been fully understood. Hence, an attempt has been made to collect data from Entrez Genome View, Cancer Genome Anatomy Project (NCBI) and Online Mendelian Inheritance in Man (OMIM) databases for enumerating and locating the number of SNPs in the loci of various chromosomes involved in cancerous and non-cancerous breast tissue. DNA methylation and mutations in the hotspots of the proto-oncogenes leading to formation of carcinomas in the breast tissue have been identified and discussed. Transition in the hotspots may reflect endogenous mutations and sporadic loss of DNA repair factors. This has been related to various molecular mechanisms and gene expression variations. SNPs also play important roles in conformational changes in proteins, resulting in non-functional or truncated proteins at the post-translational levels. Although SNPs in introns may not directly result in a change in function of a gene, they may be taken as biomarkers to locate the site near genes. SNPs technology has enormous potential in clinical research and pharmacogenomics. Hence, effort has been to understand the basic difference between candidate and validated SNPs and their significance in carcinogenesis or predisposition.

Keywords: SNPs, breast cancer genes, loci, DNA-repair, pharmacogenomics

IPC Code: Int.Cl.8 C12N15/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 463-468

 

 

Preparation and in vitro cytotoxic evaluation of taxol immunoconjugates

 

K Prabhakar, S Ganesh, V Baheti, S Saisivam, Y Madhusudan Rao and V Kishan*

Department of Pharmaceutics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506 009, India

Received 17 April 2006; revised 27 December 2006; accepted 25 February 2007

The taxol (paclitaxel) immunoconjugates prepared by derivatizing the taxol at two positions and linking the Mab CIBCNSH3 against EGF receptors were tested for in vitro cytotoxicity. Succinyl derivatives of taxol at 2˘-OH and 7-OH groups were prepared as per the published procedures. Both the products were purified by column chromatography. The
7-succinyl taxol and 2
˘-succinyl taxol immunoconjugates were prepared with CIBCNSH3, Mab against EGF receptor using a published procedure involving water soluble EDC. The unbound taxol derivatives were separated either by dialysis or centricon separators. The drug to antibody ratio was estimated by either extracting the linked drug after hydrolysis of a conjugate or simultaneous Beer’s law. The Drug-immunoconjugates were tested for their in vitro cytotoxic effect on EGF receptor expressing cells either A-549 or MCF-7 and HBL-100 by using MTT protocol and the IC50 values were calculated. The drug to antibody ratio was 0.45:1 for 7-succinyl taxol, and 0.38:1 for 2˘-succinyl taxol. The IC50 value was 78 nM on
A-549 cells for 7-succinyl taxol conjugate, while for 2
˘-succinyl taxol conjugate the IC50 values were 114.8 nM and 120.3 nM on MCF-7 and HBL-100 cells respectively. Overall, as compared to the pure antibody both taxol immunoconjugates showed more cytotoxic activity.

Keywords: taxol immunoconjugates, succinyl taxols, Mab conjugates, drug immunoconjugates, drug targeting and anticancer conjugates

IPC Code: Int. Cl.8 A01N63/00; C12N15/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 469-478

 

 

Statistical evaluation of medium components by Plackett-Burman experimental design and kinetic modeling of lipase production by Pseudomonas fluorescens

Aravindan Rajendran*, Meikandhan Thirugnanam and Viruthagiri Thangavelu

Biochemical Engineering Laboratory, Department of Chemical Engineering

Annamalai University, Annamalainagar 608 002, India

Received 8 August 2006; revised 8 February 2007; accepted 10 April 2007

The evaluation of medium components for lipase production by Pseudomonas fluorescens in submerged batch fermentation was studied using Plackett-Burman experimental design. Twelve medium components with three dummy variables were studied in this experimental design. The most significant variables affecting lipase production were found to be glucose, olive oil, yeast extract, dipotassium hydrogen phosphate and ferrous sulphate heptahydrate. Maximum lipase activity of 3.32 U mL-1 and maximum cell mass concentration of 2.15 g L-1 was obtained in the 32 h of fermentation using the optimized medium under optimized conditions of 30°C, with an initial pH of 7.0 at 120 rpm. Various unstructured kinetic models were analyzed to simulate the experimental values of cell growth, lipase activity and glucose concentration.  Logistic model for cell growth, Luedeking-Piret model for lipase production and modified Luedeking-Piret model for substrate utilization were found to predict the fermentation profile more accurately with high determination coefficient (R2) values of 0.9893, 0.9314 and 0.9765, respectively. The estimated values of the kinetic model parameters, α and β for lipase production clearly indicate that the lipase production by P. fluorescens is growth-associated.

Keywords: Pseudomonas fluorescens, submerged fermentation, lipase, Plackett-Burman experimental design, unstructured kinetic modeling

IPC Code: Int. Cl.8 C12N9/20

Indian Journal of Biotechnology

Vol 6, October 2007, pp 479-484

 

 

Covalent immobilization of lipase onto onion membrane affixed on plastic surface: Kinetic properties and application in milk fat hydrolysis

Vikas, R Kumar and C S Pundir*

Biochemistry Research Laboratory, Department of Biosciences, M D University, Rohtak 124001, India

Received 10 October 2005; revised 7 November 2006; accepted 20 February 2007

Lipase from porcine pancreas was immobilized covalently onto onion membrane, affixed on plastic surface through non-reactive fixative, with a conjugation yield of 0.174 mg/cm2 and 63.6 % retention of initial activity of free enzyme. The enzyme showed no change in optimum pH 7.5 but slight change in incubation temperature from 35 to 40şC and time of linearity from 15 to 30 min after immobilization. The utility of membrane bound lipase to prepare fat free milk without degradation of other essential components and formation of unwanted by-products was tested employing commercial kit for TG determination. Thus, the use of immobilized lipase in hydrolysis of milk fats increases the digestion process with out consuming them. The immobilized enzyme was reused 100 times without considerable loss of activity, when stored at 4şC for a period of 2 months.

Keywords: Lipase, porcine pancreas, onion membrane, immobilization, covalent coupling, milk, triglyceride.

IPC Code: Int. Cl.8  C12N9/20, 11/08

Indian Journal of Biotechnology

Vol 6, October 2007, pp 485-489

 

 

 

Homology modeling of putative thioredoxin from Helicobacetr pylori

 

D Premalatha, P Ravindra1 and L Venkateshwar Rao*

Department of Microbiology, University College of Science, Osmania University, Hyderabad 500 007, India

1School of Engineering and Information Technology, University Malaysia Sabah, 88999 Kota Kinabalu, Sabah, Malaysia

Received 12 June 2006; revised 27 November 2006; accepted 10 January 2007

The tertiary structure of putative thioredoxin (trx) of Helicobacter pylori was generated based on structural homology of the X-ray crystallographic structure of thioredoxin from Escherichia coli. Inspection of theoretically predicted structure indicates that the thioredoxin of H. pylori is similar to that of E. coli. Analysis of the structure revealed that thioredoxins have a common fold, characterized by a core of twisted β-pleated sheet flanked either side by helices. The amino terminal end of the molecule is occupied by β-α-β motif and carboxy terminal end by β-β-α motif. This molecule is characterized by five strands and four helices. Among the four helices, α2 is the longest helix which was disrupted near proline. Proline72 is identified as cis-proline. This structure retained overall trx-fold with the conservation of global shape and the secondary structures. This work determines the structure of thioredoxin and is found to be unique for further insight into molecular characterization.

Keywords: BLAST, PDB, thioredoxin, RMSD

IPC Code: Int. Cl.8 G01N33/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 490-494

 

 

Charcterization and screening of beneficial bacteria obtained on
King’s B agar from tea rhizosphere

 

Tanushree Mazumdar1, C Goswami1 and N C Talukdar2*

1Soil Microbiology Laboratory, Department of Soil Science, Assam Agricultural University, Jorhat 785 013, India

2Microbial Resources Division, Institute of Bioresources and Sustainable Development
Department of Biotechnology (Government of India), Imphal 795 001, India

Received 13 June 2005; revised 18 December 2006; accepted 22 February 2007

Nine fluorescent Pseudomonas isolates obtained on King’s B agar from rhizosphere of tea plants were studied along with a reference strain P. fluorescens MTCC-103 for their biochemical and functional characteristics. They were also tested for their ability to promote growth of tea seedlings. Intrinsic antibiotic resistance profile (IARP) and SDS-PAGE banding pattern indicated that the isolates were distinct from each other and these two properties could be used as marker for their identification. The isolates produced IAA-like substances, siderophores and soluble P in the range of 8.7-32.1, 13.6-196.3 and 1.4-15.7 µg/mL culture filtrate, respectively. Four isolates were able to utilize cellulose as C-source and another four were capable of inhibiting growth of the saprophytic Rhizoctonia solani in laboratory bioassay. The isolates Psd 11, Psd 12 and Psd 13 were found to enhance total shoot and root biomass of 1-year-old tea seedlings, grown in a fertilizer unamended soil, to significantly greater magnitude. Psd 11 and Psd 13 were identified as two different strains of Bacillus circulans. The growth parameters of tea seedlings in fertilizer P added pot was statistically at par with those of superior strain inoculated seedlings. There was no statistically significant relationship between in vitro determined functional properties observed and the growth promotion ability of the isolates. Although, the three superior isolates were found to produce higher level of soluble P and exhibited maximum in vitro biocontrol activity against R. solani.

Keywords: Bacillus, IAA, P solubilization, Pseudomonas, siderophore, tea growth promotion

IPC Code: Int. Cl.8 C12N15/10

Indian Journal of Biotechnology

Vol 6, October 2007, pp 495-503

 

 

Isolation of pigeon pea (Cajanus cajan L.) legumin gene promoter and identification of conserved regulatory elements using tools of bioinformatics

Rajani Jaiswal1, Vikrant Nain1, M Z Abdin2 and P A Kumar1*

1NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110 012, India

2Centre for Transgenic Plants Development, Department of Biotechnology, Jamia Hamdard, New Delhi 110 062, India

Received 5 January 2006; revised 28 October 2006; accepted 25 January 2007

A seed specific legumin gene promoter from pigeon pea was isolated by PCR amplification. Database assisted sequence analysis of this promoter revealed several putative cis-acting regulatory elements. Comparative analysis of 15 seed-specific legumin gene promoters from six species, viz. Cajanus cajan, Cicer arietinum, Pisum sativum, Glycine max, Vicia faba and Arachis hypogaea, revealed several conserved motifs in promoter sequences; maximum conservation was observed upstream to transcription start site. Most of the conserved motifs have known transcription factor binding sites. One unknown conserved motif of seven base pair (AG/TGTGTA) was found 19 bp upstream to legumin box, putatively named as L-19. Study of nucleosome formation potential showed that putative linker DNA is more prone to mutations as compared to DNA involved in nucleosome formation. A chimeric construct was made with pigeonpea legumin promoter and β-glucuronidase (GUS) gene. Analysis of GUS expression at different developmental stages of transgenic tobacco plant’s parts revealed that the reporter gene was expressed at a high level only in mature seeds, specifically in embryo, endosperm and in cotyledonary leaves of developing seedling. These data showed that GUS gene transcription was regulated in a tissue specific and temporally regulated manner.

Keywords: β-glucuronidase (GUS), legumin gene, nucleosome positioning, phylogenetic footprinting

IPC Code: Int. Cl.8 C12N15/09, 15/29

Indian Journal of Biotechnology

Vol 6, October 2007, pp 504-507

 

 

Biolistic-mediated DNA delivery and transient expression of GUS in hypocotyls of Feronia limonia L.—A fruit tree

S D Purohit*, S Raghuvanshi1 and A K Tyagi1

Plant Biotechnology Laboratory, Department of Botany, Mohanlal Sukhadia University, Udaipur 313 001, India

1Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110 021, India

Received 2 May 2006; revised 22 December 2006; accepted 6 March 2007

Feronia limonia L. (Rutaceae) is an important fruit tree of arid horticulture. To be able to introduce genes of interest for its improvement, biolistic-mediated DNA delivery method has been standardized. The hypocotyls explants were bombarded with plasmid pBI121 having gus reporter gene driven by CaMV 35S promoter and npt II under control of nos promoter as selectable marker. The best transient expression of GUS was recorded when tungsten particles coated with DNA were bombarded at a disc pressure of 1350 and 1100 psi from a distance of 9 cm. Some of the bombarded explants are being maintained to obtain viable regenerates for their analyses.

Keywords: biolistic, Feronia limonia, transient expression

IPC Code: Int. Cl.8 A01H15/00; C12N15/09, 15/11

Indian Journal of Biotechnology

Vol 6, October 2007, pp 508-518

 

 

Effect of salinity on growth and proteomic changes in two cultivars of mulberry (Morus alba L.) with contrasting salt tolerance

G Jyothsna Kumari, S Giridara Kumar2, M Thippeswamy, A Annapurnadevi1

S Thimma Naik and Chinta Sudhakar1*

Departments of Botany and 1Biotechnology, Sri Krishnadevaraya University, Anantapur 515 003, India

2 Department of Plant and Soil Sciences, Mississippi State University, Mississippi, MS-39762, USA

Received 18 May 2006; revised 15 February 2007; accepted 15 April 2007

The effect of salinity on growth and proteomic changes was studied in two cultivars of mulberry (Morus alba L.) with contrasting salt tolerance. Three-month-old mulberry plants were subjected to 0.0 (control), 1.0, 1.5 and 2.0% NaCl stress for 7 d. Salt treatment did affect the growth, dry matter and leaf area in both the cultivars. The cultivar S1 showed a lesser rate of decrease in growth, dry matter and leaf area than cultivar ATP which indicated that cultivar S1 is better tolerant than cultivar ATP to salt stress. Soluble proteins extracted from leaves were analyzed by two-dimensional electrophoresis in order to detect salt stress induced changes in the polypeptide patterns. Salt stress resulted in differential changes in protein level qualitatively and quantitatively in both the cultivars. More than 150 protein spots that were detected in leaves of both cultivars showed reproducible abundance within replications and significant response to salt treatment compared to unstressed. In cultivar S1, 33 proteins increased and 21 proteins decreased by different salt stress levels. Among these, protein spot # 10, 32, 33, 46 to 48 and 53 were specifically induced by NaCl treatments in S1 cultivar. In ATP cultivar, eight proteins increased and 43 proteins decreased and one protein spot (# 55) found to be specific to this cultivar. In general, salt stress resulted a much decrease in growth and total polypeptide profiles in the susceptible cultivar ATP, than tolerant cultivar S1.

Keywords: Morus alba, mulberry, stress-proteins, salt stress tolerance, two-dimensional electrophoresis

IPC Code: Int. Cl.8 G01N33/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 519-524

 

 

Assessment of genetic diversity in Azadirachta indica based on
DNA fingerprinting

R S Dhillon1*, T Mohapatra2, S Singh3, K S Boora4 and K Singh1

1Departments of Forestry, 3Genetics and 3Biotechnology & Molecular Biology, CCS Haryana Agricultural University
Hisar 125 001 India

2National Research Centre on Plant Biotechnology, IARI, New Delhi 110 012, India

Received 3 May 2006; revised 9 February 2007; accepted 5 April 2007

RAPD molecular markers were used to evaluate the genetic diversity in populations of Azadirachta indica A. Juss (neem) from different eco-geographical regions of India. Out of the 40 decamer primers used, 24 yielded polymorphic banding patterns. In total, 152 different DNA bands were reproducibly obtained, out of which 104 (68.4%) were polymorphic. The polymorphisms were scored and used in band-sharing analysis to identify genetic relationships. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA grouped all the 36 populations into two major groups. Similarity indices ranged from 0.60 to 0.94. The highest similarity coefficient detected between candidate plus trees from Jodhpur and lowest in the populations of Kalka and Banaswara, indicated that neem germplasm within India constitutes considerably broad genetic base. Also, the neem populations from diverse agroclimatic regions of India were more dispersed on the principal coordinates plot, revealing a wide genetic base.

Keywords: Azadirachta indica, neem, genetic variation, candidate plus trees, RAPD

IPC Code: Int. Cl.8 C12N15/10, 15/11

Indian Journal of Biotechnology

Vol 6, October 2007, pp 525-531

 

 

Optimization of guggulsterone production in callus cultures of Commiphora wightii (Arnott.) Bhandari

Meeta Mathur, A K Jain, S Dass and K G Ramawat

Laboratory of Bio-Molecular Technology, Department of Botany, M L Sukhadia University, Udaipur 313 001, India

Received 18 May 2006; revised 1 February 2007; accepted 8 April 2007

Commiphora wightii (Arnott.) Bhandari is an endangered, slow growing medicinal tree. Its hypolipidemic and hypocholesterolemic activity is due to the presence of two closely related steroidal ketones guggulsterone-E and guggulsterone-Z in the resin. The callus cultures derived from zygotic embryos and leaf explants were exploited for the optimal production of these bioactive molecules. The production of guggulsterone in callus cultures was maximal during
35-d growth on modified MS medium. A correlation has been observed between guggulsterone yield in callus and in vivo in the plant. Guggulsterone content of callus was maximum during January to July, the period of gum exudation in nature. The cultures, which were grown on modified MS medium (950 mg/L KNO3, 825 mg/L NH4NO3 and 220 mg/L CaCl2.2H2O) containing 2, 4, 5-T (0.25 mg/L) and kinetin (0.1 mg/L) (referred to as CM2 medium) accumulated ~15 µg/g guggulsterone. The optimal level of nitrogen [NH4NO3 (1650 mg/L), KNO3 (475 mg/L)], CaCl2 (110 mg/L) KH2PO4 (170 mg/L) in different media resulted in maximum accumulation of total guggulsterone up to 19 µg/g dry weight basis. The significant increase in total guggulsterone accumulation (59 µg/g) was recorded in the tissues grown on CM2 medium containing 40 g/L maltose. Significant increase in guggulsterone content was recorded in the tissues grown on the production medium with either sucrose or maltose alone (40 g/L) or glucose:sucrose ratio (20:20 g/L). Combination of all the optimal concentrations of salts and sugars did not further increase the guggulsterone accumulation. It appears that guggulsterone production reached to its maximum possible level (~3 fold increase) with medium salt manipulation containing 40 g/L maltose or glucose: sucrose (~60 μg/g dry weight) and further increase was not possible within these conditions

Keywords: Commiphora wightii, guggulsterones, callus cultures, optimization

IPC Code: Int. Cl.8 A01H4/00; A61P3/06

Indian Journal of Biotechnology

Vol 6, October 2007, pp 532-540

 

 

Plant regeneration in different genotypes of indica rice

 

Anusrita Biswas and Asit B Mandal*

Biotechnology Section, Central Agricultural Research Institute, Port Blair 744101, India

Received 23 September 2005; revised 12 September 2006; accepted 10 January 2007

Of 10 indica rice varieties assessed, Annada showed the best in vitro culture response in respect of callus induction, plantlet regeneration and number of plantlets/seed callus. Other high yielding varieties, viz. Kasturi, IR 72 and Karnal Local, also displayed appreciable performance. Callus induced and proliferated in dark and gave high plantlet regeneration and more plantlets/seed callus across genotypes. Among three shock treatments, viz. heat (37°C for overnight), cold (4°C for over night) and dehydration, dehydration significantly enhanced the plantlet regeneration and number of plantlets/seed callus. Of 3-dehydration treatments (6, 12 and 24 h duration), the maximum plantlet regeneration and number of plantlets/seed callus were observed in the calli dehydrated for 12 h. Of three tested media, viz. MS, LS, and N6, MS emerged the best for callus induction, plantlet regeneration and number of plantlets/seed callus. In contrast, among the modified media formulations, modified N6 [5 mM (NH4)2 SO4] produced maximum plantlets/seed callus across varieties. Increased (5 mM) (NH4)2SO4 content in the medium was found to be better than the higher dose of KNO3 or its normal level in the respective media formulations. Across genotypes, 2 mg L-1 2,4-D in the callus induction medium (CIM) was the most suitable for callus induction. However, 2,4-D in combination with other growth regulators, such as 2 mg L-1 2,4-D and 1 mg L-1 Kn, performed better. In regeneration medium (RM), 2 mg L-1 BAP with 1 mg L-1 Kn and 0.5 mg L-1 NAA, or 2 mg L-1 BAP with 0.5 mg L-1 NAA, were optimum for the maximum plantlet regeneration. Across varieties, coconut water (10%) was found to be the most effective to enhance the in vitro culture response as evident from prolific callus induction, increased plantlet regeneration and more plantlets/seed callus.

Keywords: Genotypes, rice, media, adjuvant, hormones

IPC Code: Int. Cl.8  A01H4/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 541-544

 

 

In vitro propagation of an endemic umbellifer, Hydrocotyle conferta

 

S Karuppusamy, V Aruna, C Kiranmai and T Pullaiah*

Departments of Biotechnology and *Botany, Sri Krishnadevaraya University, Anantapur 515 003, India

Received 19 April 2006; revised 6 February 2007; accepted 5 April 2007

A protocol for in vitro propagation of Hydrocotyle conferta Wight (Apiaceae) through axillary bud multiplication was established. Murashige and Skoog (MS) medium with 6.66 mM N6-benzyladenine (BA) and 5.37 mM a-naphthalenacetic acid (NAA) was best suited for axillary bud multiplication including a mean of 21 shoots/node. Excision and culture of the nodal segments from the in vitro shoots on fresh medium with same concentrations of BA and NAA facilitated development of more than 25 shoots/node. Subsequent cultures enhanced the rate of shoot proliferation. The developed shoots rooted best on half strength MS medium with 0.54 mM NAA. Plantlets established in pots exhibited 95% survival.

Keywords: axillary bud multiplication, Hydrocotyle conferta, endemic umbellifer

IPC Code: Int. Cl.8 A01H4/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 545-548

 

 

An alternative source for regenerable organogenic callus induction in
Jatropha curcas L.

Shilpa Rajore1* and Amla Batra

Biotechnology Laboratory, Department of Botany, University of Rajasthan, Jaipur 302 004, India

*1Vedic Balika P G Mahavidyalaya, Varun Path, Mansarovar, Jaipur 302 020, India

Received 27 July 2006; revised 12 February 2007; accepted 15 April 2007

Plant regeneration of Jatropha curcas was achieved through organogenesis in callus cultures. Calli were induced from leaf explants on MS basal medium supplemented with NAA (1.0 mg/L) and BAP (5.0 mg/L). Green compact nodules containing clusters of meristematic centres were induced in these calli after transfer to MS basal medium containing various concentrations of BAP/Kn (0.5-5.0 mg/L) alone or in combination with auxins. MS medium supplemented with BAP (1.5 mg/L) and IBA (0.5 mg/L) was found to be the most effective combination for shoot bud differentiation. The microshoots rooted well on MS+IBA (3.0 mg/L) medium and the plantlets successfully acclimatized in soil.

Keywords: plant regeneration, organogenesis, Jatropha curcas, agro-forestry

IPC Code: Int. Cl.8 A01H4/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 549-552

 

 

Protocol optimization for RAPD in cyanobacteria

Shalini, Saurabh Tiwari, Rajinder K Gupta!, Sunil Pabbi & Dolly Wattal Dhar*

Centre for Conservation and Utilisation of Blue Green Algae, IARI, New Delhi 110 012, India

1School of Biotechnology, Guru Gobind Singh Indraprasatha University, Delhi 110 006, India

Received 28 September 2006; revised 29 January 2007; accepted 26 March 2007

Random amplification of polymorphic DNA (RAPD) analysis using the polymerase chain reaction proved a useful technique in the biodiversity analysis of microorganisms but may lack reproducibility in the poorly standardized methodology. In the present investigation, the RAPD technique was optimized for characterizing cyanobacterial isolates in order to ensure its reproducibility and discriminatory power. Cyanobacterial isolates from the three genera, Anabaena, Nostoc and Calothrix were examined for the fragment patterns produced using different 10-mer RAPD primers and concentrations of different components (DNA template, primer and Taq polymerase) at two different annealing temperatures. It was observed that variations associated with all the parameters tested modified the fingerprinting pattern. For conditions not optimized, RAPD bands were faint and difficult to score. Therefore, a set of conditions for RAPD-PCR reaction has been defined, to ensure simple and fast reproducibility.

Keywords: optimization, RAPD-PCR, cyanobacteria

IPC Code: Int.Cl.8 C12N115/10

Short Communications

Indian Journal of Biotechnology

Vol 6, October 2007, pp 553-556

 

 

Enzymatic characteristics of lignin peroxidases of indigenous lignolytic fungal strains — Part I

 

V K Patel, R S S Yadav and K D S Yadav*

Department of Chemistry, D D U Gorakhpur University, Gorakhpur 273 009, India

Received 29 August 2005; revised 13 November2006;
accepted 23 March 2007

Extracellular production of lignin peroxidases from six indigenous fungi, viz. Abortiporus biennis MTCC-1176, Pestalotia bicolor MTCC-372, Heterobasidium annosum MTCC-146, Pleurotus ostreatus MTCC-142, Gloephyllum striatum MTCC-1117 and Loweporus lividus MTCC-1178, in liquid culture growth medium amended with lignin-containing natural substances and their enzymatic characteristics were studied. P. ostreatus MTCC-142 was found to be the best secretor of lignin peroxidase (3.50 IU/mL) in the presence of saw dust. Moreover, the enzymatic characteristics (Km, pH and temperature optima) of the lignin peroxidases produced by indigenous fungal strains were in the same range as the enzymatic characteristics of Phanerochacte chrysosporium ATCC-24725, an American type culture collection widely used for purification of lignin peroxidase. Thus, P. ostreatus MTCC-142 has potential to be used for commercial production of lignin peroxidase

Keywords: lignin peroxidase, lignin-containing natural substances, lignolytic fungi, Pleurotus ostreatus, Phanerochacte chrysosporium

IPC Code: Int. Cl.8 A01G1/04; A01N63/04

Indian Journal of Biotechnology

Vol 6, October 2007, pp 557-559

 

 

Sericulture pupal waste — A new production medium for mass cultivation of
Bacillus thuringiensis

 

A Karthikeyan and N Sivakumar*

P G and Research Department of Microbiology
J J College of Arts and Science, Pudukkottai 622 404, India

Received 27 December 2005; revised 14 February 2007;
accepted 15 March 2007

An attempt has been made to utilize the silkworm pupal waste for mass cultivation of biopesticide bacterium, Bacillus thuringiensis. The mass production potential was tested in a semi solid state fermentation set-up and the dry pupal waste powder was analyzed for carbon, lipid, protein and pH before and after solid state fermentation, where viable spore count (VSC) was taken as a criteria for evaluating the efficiency of pupal waste medium. A very high VSC of 369´109 was obtained in the present investigation.

Keywords: Bacillus thuringiensis, Bombyx mori, silkworm, sericulture pupal waste, production medium

IPC Code: Int. Cl.8 C12N1/16

Indian Journal of Biotechnology

Vol 6, October 2007, pp 560-563

 

 

Molecular identification of Proutista moesta as the vector and the phylogenetic analysis of KWD phytoplasma

Boby T Edwin and C Mohankumar*

Plant Biochemistry and Molecular Biology Research Lab Department of Botany, University College
Thiruvananthapuram 695 034, India

Received 10 January 2006; revised 27 November 2006;
accepted 8 February 2007

The purified genomic DNA from the tissues of Proutista moesta and Stephanitis typica, considered as possible vectors of Kerala wilt disease (KWD) of coconut palm, was subjected to PCR assay using the primer pairs P1/P6, P1/P7 and P4/P7. In case of P. moesta, the amplified products resolved a prominent band of 650 bp for the universal primer pair P4/P7; however, no bands were noticed for the primer pairs P1/P6 and P1/P7. In case of
S. typica no bands were noticed for all sets of primers. Since P4/P7 amplifies the 16S-23S intergenic spacer region of 16SrRNA gene, the 650 bp product from P. moesta indicates the presence of phytoplasma DNA. The restriction enzyme analysis of the 650 bp product, using the enzymes AluI, BclI, HindIII and RsaI, further supports the phytoplasmic nature of DNA. The presence of 650 bp product in the genomic DNA of P. moesta shows the insect being a vector of KWD phytoplasma. The sequential similarity of 650 bp of both KWD phytoplasma and the insect phytoplasma again supports the transmission of phytoplasma through the vector P. moesta. From the cladogram, it is obvious that the KWD phytoplasma is evolutionarily closest to the phytoplasma causing coconut lethal yellowing of Mexican palms within the group 16SrIV.The present study is the fist confirmed record of P. moesta as the vector of KWD by detecting KWD phytoplasma in the insect tissues by PCR based methods.

Keywords: Proutista moesta, Kerala wilt disease, phytoplasma, Universal primers, 650bp

IPC Code: Int. Cl.8 C12N15/09, 15/29

Indian Journal of Biotechnology

Vol 6, October 2007, pp 564-566

 

 

Thidiazuron induced shoot regeneration in the endangered species, Exacum travancoricum Beedi

 

P Kannan, A Premkumar and S Ignacimuthu*

Entomology Research Institute, Loyola College
Chennai 600 034, India

Received 9 March 2006; revised 31 January 2007;
accepted 2 March 2007

In vitro propagation of Exacum travancoricum through direct organogenesis with a mean of 28±0.19 shoots per internode explants was achieved upon culture on Murashige and Skoog (MS) medium with 2 mg/L thidiazuron (TDZ). Benzyladenine was inferior to TDZ in the induction of shoots. The developed shoots rooted best on MS medium with 3.0 mg/L indole-3-butyric acid (IBA). The plantlets exhibited 80% survival in ex vitro conditions.

KeywordsExacum travancoricum, plantlet regeneration, organogenesis, thidiazuron

IPC Code: Int. Cl.8 A01H4/00

Indian Journal of Biotechnology

Vol 6, October 2007, pp 567-569

 

 

Comparative studies on the digestive enzymes in the gut of earthworms, Eudrilus eugeniae and Eisenia fetida

M Lakshmi Prabha, Indira A Jayaraaj*, R Jeyaraaj and Srinivasa Rao

Departments of Biochemistry and Zoology, Kongunadu Arts and Science College, Coimbatore 641 029, India

Received 27 January 2006; revised 5 January 2007
accepted 15 March 2007

Solid waste and resource recycling are the only sensible ways to tackle the problem of municipal solid waste (MSW). The digestive enzymes and the intestinal microflora of earthworms seem to play an important role in the digestion of soil organic matter. The present investigation was aimed to study the levels of various enzymes viz. amylase, cellulase, xylanase, cellbiase, endonuclease, acid phosphatase, alkaline phosphatase and nitrate reductase and their activities in the gut of the two selected earthworms, Eudrilus eugeniae and Eisenia fetida. Overall, amylase, cellobiase, endoglucanase, acid phosphatase and nitrate reductase showed higher enzymatic activity.

Keywords: Eudrilus eugenia, Eisenia fetida, amylase, cellulase, nitrate reductase, acid phosphatase, alkaline phosphatase

IPC Code: Int. Cl.8 C05F3/00, 9/04; C12N9/20