Indian Journal of Experimental Biology

 

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VOLUME 49

NUMBER 9

SEPTEMBER 2011

CODEN: IJEB (A6) 49 (9) 641-716 (2011)

ISSN: 0019-5189 (Print); 0975-1009 (Online)

 

CONTENTS

 

Papers

 

 

 

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella Typhi Ty21a cell surface

645

Mohammed A A Sarhan, Mustaffa Musa & Zainul F Zainuddin

 

 

 

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line

654

Lovleen Saxena, Uttara Chaturvedi, Shikha Saxena, G Ravi Kumar, A P Sahoo, Sudesh Kumar, J Doley, R S Rajmani, Prafull K Singh, Rajiv Kumar & Ashok K Tiwari

 

 

 

Involvement of AMPA receptors for Mesobuthus tamulus Pocock venom-induced depression of monosynaptic reflex in neonatal rat spinal cord in vitro

660

Amar N Maurya & Shripad B Deshpande

 

 

 

Effect of nitric oxide in protective effect of melatonin against chronic constriction sciatic nerve injury induced neuropathic pain in rats

664

Anil Kumar, Seema Meena, Harikesh Kalonia, Amit Gupta & Puneet Kumar

 

 

 

Effect of combination of thalidomide and sulfasalazine in experimentally induced inflammatory bowel disease in rats

672

O Prakash, B Medhi, U N Saikia & P Pandhi

 

 

 

Effect of Zataria multiflora Bois L. on histamine (H1) receptor of guinea pig tracheal chains

679

Mohammad Hossein Boskabady & Hengameh Tabanfar

 

 

 

Effect of oleic, lauric and myristic acids on phenylephrine-induced contractions of isolated rat vas deferens

684

M L Arruzazabala, Y P駻ez, Y Ravelo, V Molina, D Carbajal, R Mas &
E Rodgriguez

 

 

 

Effect of green tea (Camellia sinensis L.) extract on morphological and functional changes in adult male gonads of albino rats

689

Amar K Chandra, Shyamosree Roy Choudhury, Neela De & Mahitosh Sarkar

 

 

 

Analgesic activity of the aqueous seed extract of Hunteria umbellata (K. Schum.) Hallier f. in rodents

698

Olufunmilayo Olaide Adeyemi, Adejuwon Adewale Adeneye & Tope Elizabeth Alabi

 

 

 

Protection against radiation clastogenecity in mouse bone marrow by
Phyllanthus niruri

704

Indu Thakur, P Uma Devi & Papiya Bigoniya

 

 

In vitro propagation and mass scale multiplication of a critically endangered epiphytic orchid, Gastrochilus calceolaris (Buch.-Ham ex J.E.Sm.) D.Don using immature seeds

711

Promila Pathak, Hossein Piri, S P Vij, K C Mahant & Shaveta Chauhan

 

 

 

Announcements

 

 

 

Interactive Workshop on Systems Approach to Modern Biomolecular Technology; 1st National Conference on Toxinology 2011 & 1st Annual Conference of Toxinological Society of India

644

 

 

______________________________

Announcements

Interactive Workshop on Systems Approach to Modern Biomolecular Technology

15-16 December 2011

Organised by the Centre of Advanced Study in Crystallography and Biophysics, and Bioinformatics Infrastructure Facility, University of Madras, the workshop will include interactive discussions on protein structure, protein function, structure determination, functional characterisation and structure-function relationships. For details, please contact, Prof. D. Velmurugan, Convenor, Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Maraimalai (Guindy) Campus, Chennai 600 025. Telephone: 044-22300122, Fax: 044-22300122, Mobile: 09841075847,
E-mail: shirai2011@gmail.com; dvelmurugan@unom.ac.in; toxinology2011@gmail.com

覧覧覧覧覧覧覧覧

1st National Conference on Toxinology 2011

&

1st Annual Conference of Toxinological Society of India

1718 December 2011

Jointly organised by the Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Poison Control Training and Research Centre, Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai the national conference will cover the following topics: Animal, microbial and plant toxins; Snake bite management; Toxins to therapeutics; Genomic analysis; Proteomic analysis; Bioinformatics and computational biology; and Molecular modelling and drug design. For further details kindly contact, Prof. D. Velmurugan, Convenor-TSICON 2011, Director & Head, Centre of Advanced Study in Crystallography & Biophysics, University of Madras, Maraimalai (Guindy) Campus,
Chennai 600 025, India. Telephone: 044-22300122, Fax: 044-22300122, Mobile: 09841075847,
E-mail: shirai2011@gmail.com; dvelmurugan@unom.ac.in; toxinology2011@gmail.com or
Prof. C. Rajendiran, Organizing Secretary TSICON-2011, Poison Control Training and Research Centre, Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai 600 003. Mobile: 09841017720, Telephone: 044-26640648, E-mail: dr.cranjendiran@gmail.com

__________________________________

 

Author Index

Adeneye Adejuwon Adewale

698

Adeyemi Olufunmilayo Olaide

698

Alabi Tope Elizabeth

698

Arruzazabala M L

684

 

 

Bigoniya Papiya

704

Boskabady Mohammad Hossein

679

 

 

Carbajal D

684

Chandra Amar K

689

Chaturvedi Uttara

654

Chauhan Shaveta

711

Choudhury Shyamosree Roy

689

 

 

De Neela

689

Deshpande Shripad B

660

Devi P Uma

704

Doley J

654

Gupta Amit

664

 

 

Kalonia Harikesh

664

Kumar Anil

664

Kumar G Ravi

654

Kumar Puneet

664

Kumar Rajiv

654

Kumar Sudesh

654

 

 

Mahant K C

711

Mas R

684

Maurya Amar N

660

Medhi B

672

Meena Seema

664

Molina V

684

Musa Mustaffa

645

 

 

Pandhi P

672

Pathak Promila

711

P駻ez Y

684

Piri Hossein

711

Prakash O

672

Rajmani R S

654

Ravelo Y

684

Rodgriguez E

684

 

 

Sahoo A P

654

Saikia U N

672

Sarhan Mohammed A A

645

Sarkar Mahitosh

689

Saxena Lovleen

654

Saxena Shikha

654

Singh Prafull K

654

 

 

Tabanfar Hengameh

679

Thakur Indu

704

Tiwari Ashok K

654

 

 

Vij S P

711

 

 

Zainuddin Zainul F

645

 

 

Keyword Index

Allodynia

664

Analgesic activity

698

Aqueous seed extract

698

 

 

Canine parvovirus

654

Chromosome protection

704

Chronic constriction injury

664

CNQX

660

 

 

D-004

684

 

 

Eukaryotic expression vector

654

 

 

FSH

689

 

 

Gamma irradiation

704

Gastrochilus calceolaris

711

Green tea

689

Guinea pig

679

 

 

Histamine receptor

679

Hunteria umbellata

698

Hyperalgesia

664

 

 

Ice nucleation protein

645

Immature seeds

711

Indian red scorpion venom

660

Inflammatory bowel disease

672

Inhibitory effect

679

 

 

LH

689

 

 

Malondialdehyde

672

Melatonin

664

Mouse bone marrow

704

Mycobacterium tuberculosis

645

Myeloperoxidase

672

 

 

Neuropathic pain

664

Non-NMDA receptors

660

Non-structural protein 1

654

NS-1 gene

654

Nutrient media

711

 

 

Oleic, Lauric and myristic acids

684

 

 

Phenylefrine induced contractions

684

Phyllanthus niruri

704

Polymerase chain reaction

654

Rodents

698

 

 

Seed germination

711

Seedling development

711

Sperm count

689

Spinal synaptic transmission

660

Sulfasalazine

672

Surface display

645

 

 

Testicular D5-3b-HSD

689

Testicular 17b-HSD

689

Testosterone

689

Thalidomide

672

Trachea

679

Tumor necrosis factor - a

672

Ty21a

645

 

 

Vas deferens

684

 

 

Zataria multiflora

679

 

 

Correspondent author has been indicated by * sign

 

 

Papers

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 645-653

 

 

Cloning, protein expression and display of synthetic multi-epitope mycobacterial antigens on Salmonella typhi Ty21a cell surface

Mohammed A A Sarhan1*, Mustaffa Musa2 & Zainul F Zainuddin3

1Department of Biology, College of Science, King Khalid University, 61413 Abha Saudi Arabia

2School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia

3Research & Innovation Division, Universiti Sains Malaysia, Penang, Malaysia

Received 9 February 2011; revised 9 May 2011

Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column. VacII gene was displayed on the cell surface of Salmonella typhi Ty21a using N-terminal region of ice nucleation proteins (INP) as an anchoring motif. Glycine method confirmed that VacII was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the
N-terminal domain INP- VacII fusion protein of the expected size (52 kDa).

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 654-659

 

 

Characterization and in vitro expression of non-structural 1 protein of canine parvovirus (CPV-2) in mammalian cell line

Lovleen Saxena, Uttara Chaturvedi, Shikha Saxena, G Ravi Kumar, A P Sahoo, Sudesh Kumar, J Doley,
R S Rajmani, Prafull K Singh, Rajiv Kumar & Ashok K Tiwari*

Molecular Biology Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute,
Izatnagar 243 122, India

Received 28 April 2011; revised 20 June 2011

Parvoviruses are small, 260-ナ-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 660-663

 

 

Involvement of AMPA receptors for Mesobuthus tamulus Pocock venom-induced depression of monosynaptic reflex in neonatal rat spinal cord in vitro

Amar N. Maurya & Shripad B. Deshpande*

Department of Physiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India.

Received 7 January 2011; revised 10 May 2011

Glutamate is a putative neurotransmitter at Ia-α motoneuron synapse in the spinal cord and mediate the action via N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. Since NMDA receptors are not involved in M. tamulus Pocock (MBT) venom-induced depression of spinal monosynaptic reflex (MSR), the present study was undertaken to evaluate the role of AMPA receptors in mediating the depression of MSR by MBT venom. The experiments were performed on isolated hemisected spinal cord from 4-6 day old rats. Stimulation of a dorsal root with supramaximal voltage evoked MSR and polysynaptic reflex (PSR) potentials in the corresponding segmental ventral root. Superfusion of MBT venom (0.3 オg/ml) depressed the spinal reflexes in a time-dependent manner. The maximum depression of MSR(~ 66%) was seen at 10 min and it was 25 min for PSR (~ 75%). The time to produce 50% depression of MSR and PSR was 6.7 ア 1.5 and 10.8 ア 2.6 min, respectively. Pretreatment of the cords with 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 0.1 μM), an AMPA receptor antagonist, blocked the venom-induced depression of MSR but not PSR. The results indicate that venom-induced depression of MSR is mediated via AMPA receptors.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 664-671

 

 

Effect of nitric oxide in protective effect of melatonin against chronic constriction sciatic nerve injury induced neuropathic pain in rats

Anil Kumar*, Seema Meena, Harikesh Kalonia, Amit Gupta & Puneet Kumar

Pharmacology division, University Institute of Pharmaceutical Sciences,
UGC Centre of Advanced Studies, Panjab University, chandigarh 160 014, India

Received 9 August 2010; revised 24 May 2011

Developing a successful treatment strategy for neuropathic pain has remained a challenge among researcher and clinicians. Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain in experimental animals. The present study was designed to explore the possible nitric oxide mechanism in the protective effect of melatonin against chronic constriction injury (CCI) of sciatic nerve in rats. Following chronic constriction injury, various behavioral tests (thermal hyperalgesia, cold allodynia) and biochemical parameters (lipid peroxidation, reduced glutathione, catalase, and nitrite) were assessed in sciatic nerves. Drugs were administered for 21 consecutive days from the day of surgery. CCI significantly caused thermal hyperalgesia, cold allodynia and oxidative damage. Chronic administration of melatonin (2.5 or 5 mg/kg, ip) significantly attenuated hyperalgesia, cold allodynia and oxidative damage in sciatic nerves as compared to CCI group. Further, L-NAME (5 mg/kg) pretreatment with sub-effective dose of melatonin (2.5 mg/kg, ip) significantly potentiated melatonin痴 protective effect which was significant as compared to their individual effect per se. However, L-arginine (100 mg/kg) pretreatment with melatonin (2.5 mg/kg, ip) significantly reversed its protective effects. Results of the present study suggest the involvement of nitric oxide pathway in the protective effect of melatonin against CCI-induced behavioral and biochemical alterations in rats.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 672-678

 

 

Effect of combination of thalidomide and sulfasalazine in experimentally induced inflammatory bowel disease in rats

O Prakasha, B Medhi a*, UN Saikiab & P Pandhia

Department of Pharmacologya and Histopathologyb, Postgraduate Institute of Medical Education and Research,
Chandigarh 160 012, India

Received 26 November 2010; revised 12 May 2011

Thalidomide provided significant protection against tri nitro benzene sulfonic acid induced colitis. Combination therapy also reduced colonic inflammation and all the biochemical parameters (myeloperoxidase assay, malondialdehyde assay and tumor necrosis factor-α, estimation) were significant as compared to control as well as thalidomide alone treated group. Combination therapy showed additive effect of thalidomide which restored lipid peroxidation as well as reduced myeloperoxidase and TNF-α towards the normal levels. Morphological and histological scores were significantly reduced in combination groups. In experimental model of colitis, oral administration of thalidomide (150 mg/kg) alone as well as its combination with sulfasalazine (360 mg/kg) significantly reduced the colonic inflammation. The results indicate the additive effect of thalidomide with sulfasalazine in rat colitis model which requires further confirmation in human studies.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 679-683

 

 

Effect of Zataria multiflora Bois L. on histamine (H1) receptor of guinea pig tracheal chains

Mohammad Hossein Boskabady* & Hengameh Tabanfar

Department of Physiology and Pharmaceutical Research Centre, School of Medicine, Mashhad University of Medical Sciences,
Mashhad, 9177948564, Iran Mehrdad Iranshahi

Biotechnology Research Centre, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

Received 29 November 2010; revised 2 May 2011

The effects of three concentrations (2.5, 5 and 10 mg/ml) of aqueous-ethanolic extract of Z. multiflora bois, 10 nM chlorpheniramine, and saline on histamine (H1) receptors were tested on two groups of guinea pig tracheal chains [trachea incubated with indomethacin (Gr. 1), and indomethacin and propranolol (Gr. 2)]. The effective concentration of histamine causing 50% of maximum response (EC50) obtained in presence of chlorpheniramine in both groups, all concentrations of the extract in group 1 and its two higher concentrations in group 2 were significantly greater than those of saline. The values of concentration ratio minus one (CR-1) obtained in presence of all the three concentrations of the extract in group 1 and 10 mg/ml concentration in group 2 were significantly greater than those of chlorpheniramine. The values of EC50 obtained in presence of all the three concentrations of extract and CR-1 obtained in the presence of 2.5 and 5 mg/ml concentrations in group 2 were lower than group 1. There was not significant difference in maximum response obtained in presence of different concentrations of extract between two groups. There were parallel right ward shift in concentration response curves obtained in presence of all concentrations of the extract in both the groups. These results indicated an inhibitory effect of Z. multiflora at histamine H1 receptors.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 684-688

 

 

Effect of oleic, lauric and myristic acids on phenylephrine-induced
contractions of isolated rat vas deferens

ML Arruzazabala, Y P駻ez, Y Ravelo, V Molina*, D Carbajal, R Mas & E Rodrguez

Centre of Natural Products, National Centre for Scientific Research

25 th Ave, 158 st., P.O 6880, Cubanac疣, Havana City, Cuba

Received 24 February 2011; revised 24 June 2011

D-004, a lipid extract of Roystonea regia fruits that contains oleic, lauric and myristic acids as major components inhibits α1-adrenoreceptors-mediated contractile responses in isolated rat vas deferens and prostate trips; no study has demonstrated a similar effect for oleic, lauric or myristic acids individually. Therefore, the effects of D-004 (250 mg/ml), oleic (100 mg/ml), lauric (50 mg/ml) or myristic (25 mg/mL) acids and their combined effects on phenylephrine (PHE: 10-7- 10-4 mol/L) induced contractions has been studied. No treatment changed the basal tone of the preparations, but all inhibited PHE-induced contractions. D-004 produced the highest inhibition, followed by lauric acid, which was more effective than myristic and oleic acids against PHE-induced contractions of control group. D-004 and the mixture of the three acids produced similar inhibitions.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 689-697

 

 

Effect of green tea (Camellia sinensis L.) extract on morphological and functional changes in adult male gonads of albino rats

Amar K Chandraa*, Shyamosree Roy Choudhurya, Neela Dea & Mahitosh Sarkarb

aEndocrinology and Reproductive Physiology Laboratory, Department of Physiology, University College of Science and Technology, University of Calcutta, 92, Acharya Prafulla Chandra Road, Kolkata 700 009, West Bengal, India

bDepartment of Physiology, Gurunanak Institute of Dental Science and Research, Kolkata, 700 114, India

Received 17 March 2011; revised 21 June 2011

Green tea, prepared from the steamed and dried leaves of the shrub Camellia sinensis, is known for its antioxidant and anti-carcinogenic effects. However, its effects on male gonadal functions have not been explored adequately and the present investigation has been undertaken to evaluate the effect of green tea extract on gonads of adult male albino rats. Results of in vivo studies showed that green tea extract (GTE) at mild (1.25 g%, ≡ 5 cups of tea/day), moderate (2.5 g%, ≡ 10 cups of tea/day) and high (5.0 g%, ≡ 20 cups of tea/day) doses, for a period of 26 days, altered morphology and histology of testis and accessory sex organs. A significant dose-dependent decrease in the sperm counts, inhibited activities of testicular ∆53β- and 17β-hydroxysteroid dehydrogenase (∆5-3β-HSD and 17β-HSD respectively) and decreased serum testosterone level were noticed. Significant increase in serum LH level was observed after moderate and high doses; serum FSH level also increased but not significantly. Histopathological examination showed inhibition of spermatogenesis evidenced by preferential loss of matured and elongated spermatids. Results of this study showed that GTE at relatively high dose may cause impairment of both the morphological and normal functional status of testis in rodents and thus its consumption at relatively high doses raises concern on male reproductive function in spite of its other beneficial effects.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 698-703

 

 

Analgesic activity of the aqueous seed extract of Hunteria umbellata (K. Schum.) Hallier f. in rodents

Olufunmilayo Olaide Adeyemi1,*, Adejuwon Adewale Adeneye1,2,* & Tope Elizabeth Alabi1

1Department of Pharmacology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos,
Idi-Araba, Lagos State, Nigeria

2Department of Pharmacology, Faculty of Basic Medical Sciences, Lagos State University College of Medicine,
Ikeja, Lagos State, Nigeria

Received 6 April 2011; revised 24 June 2011

The analgesic effect and possible mechanism(s) of action of 50-200 mg/kg of the aqueous seed extract of H. umbellata (HU) were investigated in different experimental models of analgesia using the tail flick, tail immersion, acetic acid-induced writhing tests and formalin-induced algesia. Oral pre-treatment with 50-200 mg/kg of HU caused significant and dose related analgesic effect in the treated rats in all the experimental models used. This analgesia was mediated via central and peripheral mechanisms. Overall, the results showed that HU possesses analgesic effect which lends support to its folkloric use in the local management of pain.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 704-710

 

 

Protection against radiation clastogenecity in mouse bone marrow by
Phyllanthus niruri

Indu Thakur1, P Uma Devi1, & Papiya Bigoniya2*

1Department of Research, Jawaharlal Nehru Cancer Hospital and Research Centre, Idgah Hills, Bhopal 462002, India

2Department of Pharmacology, Radharaman College of Pharmacy, Ratibad, Bhopal 462002, India

Received 11 January 2011; revised 31 May 2011

The effects of aqueous (PnAq) and alcoholic (PnAl) extract (50-250 mg/kg) of P. niruri on in vivo gamma radiation induced chromosome aberration and in vitro antioxidant activity (50-500 オg/ml) were studied. The antioxidant activity was studied by measuring inhibition of hydroxyl radicals generated by the fenton reaction along with pro-oxidant and iron chelating ability. PnAl showed highly significant in vitro free radical scavenging ability when compared to DMSO above 250 オg/ml concentration. PnAq showed significant pro-oxidant activity while PnAl was devoid of it at the tested concentrations. Exposure to gamma radiation (4 Gy) caused 29.10 % increase in the frequency of chromosomal aberrations. Administration of PnAl (250 mg/kg) showed highly significant decrease in chromosomal aberrations compared to radiation treated group. Radioprotective potential of alcoholic extract was found to be more effective than the aqueous extract. Qualitative phytochemical investigation of PnAq and PnAl revealed the presence of sugars, flavonoids, alkaloid, lignans, polyphenols, tannins, coumarins and saponins. Higher radioprotective effect of the alcoholic extract may be attributed to rich presence of antioxidant polyphenolic compounds.

 

 

 

Indian Journal of Experimental Biology

Vol. 49, September 2011, pp. 711-716

 

 

In vitro propagation and mass scale multiplication of a critically endangered epiphytic orchid, Gastrochilus calceolaris (Buch.-Ham ex J.E.Sm.) D.Don.
using immature seeds

Promila Pathak*, Hossein Piri, S P Vij, K C Mahant & Shaveta Chauhan

Orchid Laboratory, Department of Botany, Panjab University, Chandigarh 160 014, India

Received 24 May 2010; revised 20 May 2011

In vitro asymbiotic seed germination potential of its immature seeds (36 weeks after pollination) of G. calceolaris was successfully tested on three different agar gelled nutrient media i.e. Murashige and Skoog (MS), Mitra et al. (M) and potato dextrose agar (PDA). Seeds germinated within 15.75ア0.75 to 35.75ア0.75 days in the three different media. The protocorms developed therefrom subsequently differentiated into first leaf and root primordia, and complete seedlings were obtained within 111.25ア1.25 to 141.25ア1.25 days on MS and M media. The protocorms, though failed to differentiate further on basal PDA medium, despite repeated subculturings, incorporation of peptone (P; 1gl-1), yeast extract (YE; 2 gl-1) and coconut water (CW; 20%) in the medium proved beneficial in inducing differentiation, in these germinating entities. Additional use of growth additives (P/YE/CW), in general, favoured better germination, protocorm formation and seedling development. The optimal nutritional combination during seed germination, protocorm growth and multiplication and seedling development was found to be CW (10%) enriched MS medium.