Indian Journal of Biotechnology

 

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VOLUME 7

CODEN: IJBNAR 7(2) (2008) 151-276

NUMBER 2

APRIL 2008

ISSN: 0972-5849

 

 

CONTENTS

 

Reviews

 

Biosorption of heavy metals–An overview

159

Nilanjana Das, R Vimala & P Karthika

 

Prospects of lactic acid bacteria of marine origin

170

K Kathiresan & G Thruneelakandan

 

Stem cell based therapy to restore nearly normal hearing

178

K Ananda Krishna & K R S Sambasiva Rao

 

Papers

 

Characterization and genomic organization of esterase gene in silkworm, Bombyx mori L.

183

K M Ponnuvel, K Ashok Kumar, D Velu, P Somasundaram, R K Sinha & C K Kamble

 

Molecular evaluation of bivoltine, polyvoltine and mutant silkworm (Bombyx mori L.) with
RAPD, ISSR and RFLP-STS markers

188

A K Awasthi, P K Kar, P P Srivastava, Nidhi Rawat, K Vijayan, A R Pradeep & S Raje Urs

 

Comparison of nucleotide and amino acids sequence of Nilgai (Boselaphus tragocamelus)
interleukin-18 (IL-18) with other ruminants

195

Dhanjit Kumar Das, Mohini Saini, Devendra Swarup & Praveen Kumar Gupta

 

Expression of gene encoding immunodominant merozoite surface protein of Theileria annulata
in Escherichia coli

200

C Rajendran, D D Ray & G C Bansal

 

A fusion gene encoding two different insecticidal proteins of Bacillus thuringiensis

204

N Saraswathy, Vikrant Nain, K Sushmita & P Ananda Kumar

 

Molecular characterisation of traded black pepper (Piper nigrum L.) from India, Indonesia,
Vietnam and Malaysia

210

Jaleel Kizhakkayil, Elizabeth Thomas & B Sasikumar

 

Molecular characterization of Tobacco streak virus causing soybean necrosis in India

214

N Arun Kumar, M Lakshmi Narasu, Usha B Zehr & K S Ravi

 

Biotechnological production of xylitol by mutant Candida tropicalis OMV5: Process optimization using statistical approach

218

Ravella Sreenivas Rao, Cherukuri Pavana Jyothi & Linga Venkateswar Rao

 

Oxygen transfer conditions in the production of rainbow trout growth hormone (rtGH) by Escherichia coli

225

Usama Beshay

 

Thin layer chromatographic detection of poly-β-hydroxybutyrate (PHB) and poly-β-hydroxyvalerate (PHV) in cyanobacteria

230

Bhabatarini Panda, Laxuman Sharma, Akhilesh Kumar Singh & Nirupama Mallick

 

Impact of soil composting using municipal solid waste on biodegradation of plastics

235

Ch Vijaya & R Mallikarjuna Reddy

 

An integrated approach to primary and secondary hardening of banana var. Grand Naine

240

Shailesh R Vasane & R M Kothari

 

Micropropagation of Terminalia bellerica  (Roxb.) from juvenile explants

246

P Rathore, R Suthar & S D Purohit

 

Somatic embryogenesis and plant regeneration from stem explants of Leptadenia reticulata (Retz.) Wight. & Arn.

250

N Sathyanarayana, R Rajesha, P B Vikas & T N Bharath Kumar

 

Micropropagation of a rare mangrove Bruguiera cylindrica L. towards conservation

255

Varsha Vartak & Mahesh Shindikar

 

Rapid in vitro multiplication of Drosera burmanii Vahl.: A vulnerable and medicinally important insectivorous plant

260

K Jayaram & M N V Prasad

 

In vitro studies on development of gametophyte, sex-ontogeny and reproductive biology of a threatened fern, Microsorium punctatum (L.) Copel.

266

Ruchi Srivastava, Jyoti Srivastava, Sandip K Behera & P B Khare

 

Short Communication

         

Enhanced accumulation of triterpenoids and flavonoids in cell suspension cultures of Azadirachta indica (A. Juss.) with an extended stationary phase

270

Vidhu Sankar Babu, Narasimhan S & G M Nair

 

Instructions to Contributors

273

 

AUTHOR INDEX

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 159-169

 

Biosorption of heavy metals–An overview

Nilanjana Das*, R Vimala and P Karthika

School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore 632 014, India

Received 5 December 2006; revised 31 July 2007; accepted 20 October 2007

During the last two decades, extensive attention has been paid on the management of environmental pollution causal by hazardous materials such as heavy metals. Decontamination of heavy metals in the soil and water around industrial plants has been a challenge for a long time. A number of methods have been developed for the removal of heavy metals from liquid wastes such as precipitation, evaporation, electroplating, ion exchange, membrane processes, etc. However, these methods have several disadvantages such as unpredictable metal ion removal, high reagent requirement, generation of toxic sludge, etc. Biosorption is a process, which represents a biotechnological innovation as well as a cost effective excellent tool for removing heavy metals from aqueous solutions. This article provides a selective overview of past achievements and present scenario of biosorption studies carried out on some promising natural biosorbents (algae, fungi, bacteria, yeast) and some waste materials which could serve as an economical means of treating effluents charged with toxic metallic ions.

Keywords: algae, fungi, bacteria, yeast, biosorption, heavy metal, biosorbent, wastewater, biomass

 

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 170-177

 

Prospects of lactic acid bacteria of marine origin

K Kathiresan and G Thiruneelakandan

Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, India

Received 2 May 2006; revised 2 July 2007; accepted 5 October 2007

Lactic acid bacteria (LAB) are beneficial bacteria. They are known for their human gut floral maintenance, fermentative activity and food preservative capacity. The marine strains may have better potential than their terrestrial counterparts. This is due to essential nutrients provided by marine biotopes for nurturing the LAB, and to extreme environmental niches. The marine LAB and their by-products may have potential values in food processing, fermentation, pharmaceutical and biopolymer industries.

Keywords: Lactic acid bacteria, marine environment, lactobacilli, bacteriocin, probiotics

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 178-182

Stem cell based therapy to restore nearly normal hearing

K Ananda Krishna1 and K R S Sambasiva Rao*

Center for Biotechnology, Acharya Nagarjuna University, Guntur 522 510, India

Received 13 November 2006; revised 22 August 2007; accepted 20 November 2007

Hair cells with stereocilia are the sensory receptors of the inner ear that are located in the organ of corti of the cochlea, involved in detecting sound, and are connected with the nerve fibers that stretch from the inner ear to the brain. These hair cells convert sound information to electric signals, which are then sent to the higher brain centers. In humans, hair cell damage results in permanent hearing impairment; whereas, birds have the capacity to rebuild a damaged inner ear and various studies have demonstrated hair cell regeneration. Recently, stem cell research has triggered many programmes around the world to examine the possibility for regenerating the hair cells from embryonic/adult stem cells. The important spin-off of the research would be to find signaling mechanisms that regulate hair cell regeneration to restore nearly normal hearing in humans. The current review focuses on stem cell-based therapy with particular emphasis to summarize the recent progress.

Keywords: Stem cells, hair cell, cochlea

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 183-187

 

Characterization and genomic organization of esterase gene in silkworm,
Bombyx mori L.

K M Ponnuvel*, K Ashok Kumar, D Velu, P Somasundaram, R K Sinha and C K Kamble

Biotechnology Laboratory, Central Sericulture Germplasm Resources Centre, Thally Road, Hosur 635 109, India

Received 12 May 2006; revised 18 June 2007; accepted 6 October 2007

N-terminal amino acid sequence of esterase gene was blast searched with Bombyx mori EST (expressed sequence tag) database. EST clone fbpv0006 showed 95% homology with N-terminal sequence of esterase protein. Further, the genomic organization of exons and introns were identified using the EST clone sequence. The results on genomic organization of esterase gene indicated that the blood esterase gene possesses two exons with varying length of 192 and 524 bp, and a long intron of 2124 bp length present between these two exons. The primers were designed to the intron and exon regions and the fragments were PCR amplified using genomic DNA from silk moth as template. The amplicon showed a mol wt 799 bp in the intron region and 547 bp covering the exon regions. When the esterase genes from two multivoltine (Pure Mysore & PMX) and bivoltine (NB4D2 & CSR-19) races, possessing contrasting characters to thermal tolerance, were PCR amplified and products were sequenced. The two sequences showed 97% homology with 3% mismatch through pair blast analysis. Furthermore, per cent identity per exon, number of gaps per exon, overall per cent identity, per cent coverage of the mRNA, number of splice donor sites, presence or absence of splice donor and acceptor sites for each exon were inferred through spidey programme. Also, the phylogenetic relationship of B. mori esterase gene sequence was compared with other organisms through Clustal W analysis and a dendogram was projected. The projection showed that majority of insect esterases formed a major separate cluster, while B. mori esterase clustered with aphid insect esterases.

Keywords: Bombyx mori, exons, introns, BLAST, cDNA

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 188-194

 

Molecular evaluation of bivoltine, polyvoltine and mutant silkworm
(Bombyx mori L.) with RAPD, ISSR and RFLP-STS markers

A K Awasthi*, P K Kar1, P P Srivastava, Nidhi Rawat, K Vijayan, A R Pradeep and S Raje Urs

Seribiotech Research Laboratory, Central Silk Board, Carmelram Post, Kodathi, Bangalore 560 035, India

Received 29 August 2006; revised 8 August 2007; accepted 1 November 2007

Mulberry silkworm (Bombyx mori L.), the most important silk producing insect, exhibits wide diversity in morphological and biometric characters. Characterization of vast genetic resources based on the morphological and quantitative traits is not solely dependable as the phenotypic traits are influenced by environment. In order to study genetic relatedness of the selected and varied genotypes, six each of the bivoltine, polyvoltine silkworm accessions and mutant stocks, were studied with RAPD, ISSR and RFLP-STS markers. Twelve RAPD primers generated 172 markers of which 161 were polymorphic, generating 93.60% polymorphism. Pair-wise genetic divergence varied from 0.209 between Mysore Princess and Rong Diazo to 0.588 between Boropolu and TMS-35. The ISSR primers generated 156 markers of which 132 were polymorphic thus generating 84.62% polymorphism. The pair-wise genetic diversity among the genotypes varied from 0.189 between Rong Diazo and BL-23 to 0.438 between MU-10 and TMS-35. Similarly, 10 RFLP-STS primers produced a total of 69 bands, out of which 53 were polymorphic thus realizing 75.6% polymorphism. Genetic distance varied from 0.242 between Nistari-M and BL-23 to 0.730 between Fengshong and TMS-17. On clustering with UPGMA and principal component analysis (PCA), RAPD and ISSR markers clearly discriminated the bivoltines and multivoltines and a multivoltine Tamil Nadu white occupied the positions among bivoltines, since it has bivoltine parentage. Boropolu, an original land race from North East India, and Feng shong, a Chinese silkworm strain, also showed a closer genetic relationship.

Keywords: Bombyx mori, silkworm, genetic characterization, ISSR, RAPD, RFLP-STS, biovoltine, polyvoltine

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 195-199

 

Comparison of nucleotide and amino acids sequence of Nilgai
(Boselaphus tragocamelus) interleukin-18 (IL-18) with other ruminants

Dhanjit Kumar Das1, Mohini Saini1*, Devendra Swarup1 and Praveen Kumar Gupta2

1Centre for Wildlife, 2Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar 243 122, India

Received 18 April; revised 26 July 2007; accepted 10 October 2007

Interleukin-18, a component of innate immunity with potent interferon-g-inducing activity, is not well characterized in wild ruminants. In this study, IL-18 cDNA of Nilgai (Boselaphus tragocamelus) was cloned and characterized. The nucleotide sequence was 582 bp long and contained its entire open reading frame encoding 193 amino acid residues. The sequence analysis indicated seven nucleotide substitutions with reported buffalo sequence showing 98.5% similarity, 97.8% with cattle and 97.6% with sheep. The Nilgai IL-18 cDNA included a putative cleavage site of IL-1b-converting enzyme (ICE) and IL-1 signature-like sequence identified in human and cat IL-18 cDNA. Both nucleotide as well as amino acid sequence similarity shows that the cloned sequence was closer to buffalo IL-18 sequence.

Keywords: inerleukin-18, Boselaphus tragocamelus, cloning, cDNA sequence, nilgai

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 200-203

 

Expression of gene encoding immunodominant merozoite surface protein of Theileria annulata in Escherichia coli

C Rajendran, D D Ray* and G C Bansal

Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243 122, India

Received 8 January 2007; revised 6 July 2007; accepted 10 October 2007

The asexual blood stage “merozoites” of an Indian strain of the tick-borne cattle haemoprotozoa, Theileria annulata, was generated in in vitro culture and the gene encoding the merozoite surface protein (Tams 1) was amplified from cDNA by using primers designed from T. annulata (Ankara strain). The amplified gene was cloned into pPROExHT b plasmid vector and expressed as fusion protein in Escherichia coli, DH5α strain. The recombinant protein was purified using NI-NTA affinity chromatography and used to detect antibody response in known anti-Tannulata bovine serum samples in a ELISA format.

Keywords: Theileria annulata, immunodominant merozoites surface protein, pPROEXHTb, E.coli, Ni-NTA affinity chromatography, western blot analysis

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 204-209

 

A fusion gene encoding two different insecticidal proteins of
Bacillus thuringiensis

N Saraswathy, Vikrant Nain, K Sushmita and P Ananda Kumar*

National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110 012, India

Received 3 October 2006; revised 26 June 2007; accepted 15 September 2007

A chimeric fusion gene was constructed with the coding regions of insecticidal crystal protein (Cry1Ac) and vegetative insecticidal protein (Vip3Aa14) of Bacillus thuringiensis (Bt). Overexpression of the fusion gene in Escherichia coli resulted in the synthesis of a protein of ~140 kDa size, as revealed by SDS-PAGE analysis. Western hybridization analysis using polyclonal antisera raised against Cry1Ac showed the presence of a band corresponding to ~140 kDa. Stability of the fusion protein was studied by trypsin digestion. Insect bioassays of the fusion protein on three insect species viz., Helicoverpa armigera, Spodoptera litura and Plutella xylostella showed that the fusion protein retained the toxicity of Cry1Ac, but partially lost that of Vip3Aa14.

Keywords: Bacillus thuringiensis, protein engineering, vegetative insecticidal protein, transgenic plants, insecticidal crystal protein

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 210-213

 

Molecular characterization of traded black pepper (Piper nigrum L.) from
India, Indonesia, Vietnam and Malaysia

Jaleel Kizhakkayil, Elizabeth Thomas and B Sasikumar*

Division of Crop Improvement and Biotechnology, Indian Institute of Spices Research, Marikunnu P O, Calicut 673 012, India

Received 20 January 2007; revised 21 June 2007; accepted 17 September 2007

Molecular profiling (RAPD) and clustering of traded black pepper samples from India, Indonesia, Vietnam and Malaysia revealed a comparatively high genetic similarity within the samples from a particular country than between any two countries. The UPGMA dendrogram constructed based on the similarity coefficient revealed a total of four groups in two different clusters. The two Indonesian samples form cluster I, while others form cluster II. The genuine Indian varieties and the traded pepper from India formed separate group in cluster II. Similarly, black pepper from Malaysia and Vietnam also formed distinct groups in cluster II. The aspect of genetic similarity was discussed in relation to the origin and spread of black pepper.

Keywords: black pepper, India, Indonesia, Malaysia, molecular technique, pepper, Piper nigrum, Vietnam

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 214-217

 

Molecular characterization of Tobacco streak virus
causing soybean necrosis in India

N Arun Kumar1,2, M Lakshmi Narasu2, Usha B Zehr1 and K S Ravi1*

1Mahyco Research Center, Jalna-Aurangabad Road, Dawalwadi, Post Box No. 76, Jalna 413 203, India

2Center for Biotechnology, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 072, India

Received 13 March 2007; revised 3 May 2007; accepted 13 September 2007

A virus isolate infecting soybean (Glycine max L.) with characteristic symptoms of necrosis was collected from various locations of Maharashtra, India. The virus was detected as Tobacco streak virus (TSV) by direct antigen-coating-enzyme-linked immunosorbent assay (DAC-ELISA) using TSV specific antiserum. ELISA positive TSV soybean isolates upon mechanical transmission onto indicator hosts, viz. Vigna unguiculata cv. C-152, Glycine max and Nicotiana tabacum cv. Xanthi, produced characteristic local necrotic lesions on primary inoculated leaves, followed by systemic infection. Coat protein (CP) gene from a representative soybean isolate (TSV-SB) was amplified using TSV CP specific primers. The amplicon of ~750 bp was cloned and sequenced. The CP gene consists of 717 nucleotides, potential of coding a polypeptide of 238 amino acid residues. The CP gene of TSV-SB isolate shared 98.3 to 99.3% nucleotide and 96.6 to 98.3% amino acid sequence homology with the TSV isolates characterized from India. With TSV-WC (White clover isolate from America, type strain) and TSV-BR (Soybean isolate from Brazil), TSV-SB isolate shared 88.7% and 79.2% amino acid sequence homology, respectively. Phylogenetic tree constructed based on amino acid sequence analysis showed a close clustering of TSV-SB isolate with other TSV isolates from India than with the American and Brazilian strains. Based on CP gene sequence analysis, it is concluded that the TSV-SB isolate is a strain of TSV that is prevalent in India. This is the first report of molecular characterization of TSV infecting soybean from India.

Keywords: coat protein gene, Tobacco streak virus, soybean

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 218-224

 

Biotechnological production of xylitol by mutant Candida tropicalis OMV5: Process optimization using statistical approach

Ravella Sreenivas Rao, Cherukuri Pavana Jyothi and Linga Venkateswar Rao*

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

Received 22 January 2007; revised 17 August 2007; accepted 25 October 2007

An orthogonal experimental design L16 (25) was used to investigate effects of key media components namely xylose, yeast extract, peptone, urea and inoculum size on the production of xylitol by a mutant strain of Candida tropicalis (CT-OMV5). Software for automatic design and analysis of the experiments, based on Taguchi approach was used. Optimal levels of key media components were also determined. Among the tested parameters, xylose and urea contributed higher influence and yeast extract concentration also played an important role in the conversion of xylose to xylitol. Application of Taguchi method helped in easy process optimization and higher xylitol yield. The increased yield was possible at lower levels of yeast extract and peptone. The yield of xylitol under these optimal conditions was 0.89g/g of xylose.

Keywords: Candida tropicalis OMV5, mutant, orthogonal array, statistical optimization, Taguchi methodology, xylose, xylitol

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 225-229

 

Oxygen transfer conditions in the production of rainbow trout growth hormone (rtGH) by Escherichia coli

Usama Beshay*

Bioprocess Development Department, Genetic Engineering and Biotechnology Research Institute
Mubarak City for Scientific Research and Technology Applications, New Bourg El-Arab City, Alexandria, Egypt

Received 16 April 2007; revised 23 July 2007; accepted 25 September 2007

Batch cultures of Escherichia coli pRE1-rtGH 121/MZ1 were carried out at different oxygen transfer rates (OTR) enhanced by the increase of agitation and/or aeration rates. The growth kinetics of E. coli was investigated in complex medium under batch cultivation in 3 L fermenters. The agitation rates influenced both cell growth and rainbow trout growth hormone (rtGH) production in the bioreactor. It was found that increasing the agitation speed had a positive effect on the production of rtGH. Moreover, varying the airflow rate (vvm) in the fermentor under constant drive speed, cell growth and rtGH production were greatly influenced. In the production of rtGH by E. coli, high aeration rates were found to be essential for good yields of recombinant protein. Agitation speed and aeration rate could affect dissolved oxygen concentration, which in turn affected cell growth and rtGH production. Increased aeration rates induced higher rtGH production, with the highest concentration of 0.957 g/L obtained at 4.0 vvm, within 23 h. The highest volumetric productivity for rtGH of 0.042 g/L/h was obtained at both 1000 rpm and 4.0 vvm.

Keywords: aeration rate, agitation speed, Escherichia coli, fermentation, fish growth hormone

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 230-234

 

Thin layer chromatographic detection of poly-β-hydroxybutyrate (PHB)
and poly-β-hydroxyvalerate (PHV) in cyanobacteria

Bhabatarini Panda, Laxuman Sharma, Akhilesh Kumar Singh and Nirupama Mallick*

Agricultural and Food Engineering Department, Indian Institute of Technology, Kharagpur 721 302, India

Received 22 January 2007; revised 18 June 2007; accepted 25 September 2007

A simple method for the detection of poly-β-hydroxybutyrate (PHB) and poly-β-hydroxyvalerate (PHV) by thin layer chromatography was developed. Using this method, PHB and PHV were detected in four cyanobacterial species, viz. Nostoc muscorum, Spirulina platensis, Calothrix sp. and Synechocystis sp. PCC 6803. Presence of hydroxybutyrate (HB) units and hydroxyvalerate (HV) units was confirmed by UV-spectrophotometric and gas chromatographic analyses.

Keywords: Cyanobacteria, gas chromatography, β-hydroxybutyrate, β-hydroxyvalerate, propanolysis, thin layer chromatography

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 235-239

 

Impact of soil composting using municipal solid waste on
biodegradation of plastics

Ch Vijaya1 and R Mallikarjuna Reddy*

Department of Biotechnology, Jawahar Bharati Degree College, Kavali, India

1Department of Microbiology, S V University PG Centre, Kavali, 524 201, India

Received 5 February 2007; revised 20 August 2007; accepted 2 November 2007

Plastic wastes accumulating in the environment are posing an ever increasing ecological threat. In this context, an attempt was made to study the biodegradation of polyethylene films in the natural environment. The method suggested by ASTM standards, i.e. composting of polythene films with municipal solid waste, was adopted to examine the biodegradation of plastics in the natural environment. The samples were collected and tested for weight loss and reduction in tensile strength at 2 months interval for 12 months of composting. Loss of weight and reduction in tensile strength of polythene films were taken as the criteria indicating biodegradation of these materials. Composting of polythene films for 4 months did not show any degradation. After 4 months of composting, the loss in weight was 2.9-4.5% for HDPE films and 10.5-11.6% for LDPE films. Similarly, the reduction in tensile strength ranges from 16-20% for HDPE and 12-13% for LDPE films. The study indicates that the biodegradation of polythene films occur in natural environment at a very slower rate.

Keywords: Biodegradation, plastics, solid waste, soil composting, tensile strength

Indian Journal of Biotechnology

Vol 7, April 2008, pp 240-245

 

An integrated approach to primary and secondary hardening of banana var. Grand  Naine

Shailesh R Vasane and R M Kothari*

Jain Hi-Tech Agri Research Institute, Jain Irrigation Systems Ltd, Jalgaon 425 001, India

Received 17 April 2007; revised 3 August 2007; accepted 12 October 2007

For improving banana productivity, virus-indexed Grand Naine plantlets were used for primary and secondary hardening. Among nine combinations of bio-fertilizers (including VAM) used, GNP2V1 medium comprising of peat fortified with nitrogen fixing and phosphate solubilizing microbes (each 1 g/plantlet) and VAM (2 g/plantlet) emerged as the optimal growth medium for primary hardening on the basis of statistically significant 98.9% survival, besides corroborating optimal morphological features (viz. root length, number of primary roots, % VAM colonization, height, girth, number of leaves, leaf area and chlorophyll content) at the end of primary hardening. For secondary hardening, among four combinations of bio-fertilizers and VAM used in conjunction with soil, press mud cake (PMC) and vermi-compost (VC), SNP2V1 medium comprising of soil, PMC and VC in a ratio of 1:1:1 (v/v/v) emerged as the optimal growth medium on the basis of statistically significant 97% survival, corroborated by optimal morphological features as above, along with uptake of macro- and micro-nutrients. The positive feedback by several monitoring parameters have left no doubt about the integrated outcome at the end of primary and secondary hardening.

Keywords: banana, bio-fertilizers, primary hardening, secondary hardening, VAM

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 246-249

 

Micropropagation of Terminalia bellerica Roxb. from juvenile explants

P Rathore, R Suthar and S D Purohit*

Plant Biotechnology Laboratory, Department of Botany, Mohanlal Sukhadia University, Udaipur-313001, India

Received 5 January 2007; revised 20 June 2007; accepted 30 August 2007

An in vitro micropropagation system has been developed for Terminalia bellerica Roxb., an important Indian medicinal plant. Nodal segments obtained from 15-d-old aseptically grown seedlings were used as explants. MS medium containing 1.5 mg L-1 BAP was found most suitable for culture initiation. Although shoot multiplication was achieved on MS medium containing BAP and Kn, the maximum number of shoots was obtained with 1.5 mg L-1 BAP. Best rooting response (60%) was observed on medium containing quarter strength MS salts, 0.6% agar and 0.1 mg L-1 IBA. Plantlets were hardened initially in culture room conditions and then transferred to misthouse.

Keywords: Axillary bud proliferation, Bahera, in vitro, rooting, shoot multiplication

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 250-254

 

Somatic embryogenesis and plant regeneration from stem explants of Leptadenia reticulata (Retz.) Wight. & Arn.

N Sathyanarayana*, R Rajesha, P B Vikas and T N Bharath Kumar

Department of Bio-Technology, Sir M Visvesvaraya Institute of Technology, Bangalore 562 157, India

Received 28 August 2006; revised 6 June 2007; accepted 10 September 2007

Somatic embryogenesis and plant regeneration from stem explants of Leptadenia reticulata (Asclepeadaceae), an endangered medicinal plant possessing galactogogue property and rich alkaloid content, have been reported. Murashige and Skoog’s medium supplemented with MS + 3% sucrose + NAA (2.68 µM) + BAP (4.40 µM) was the best for callus formation as well as pre-embryonic mass (PEM) induction. Of the two subculture conditions tested for proper embryo induction, MS liquid medium proved superior over solid culture medium in inducing healthy embryos. Presence of NAA in the induction medium was also found to be critical for PEM formation, as the callus raised without it could not be triggered for embryogenesis in any of the subsequent subcultures. The resulting embryoids on transferring to Murashige and Skoog’s basal as well as other cytokinin containing medium attained maturation with varied frequencies, of which MS basal medium triggered maximum response compared to other cytokinin containing medium. The so-developed shoots on transferring to half strength MS + IBA (4. 90 µM) developed vigorous tap root system, which were later hardened on peat mass mixture with 75% survival.

Keywords: Stem explants, Leptadenia reticulata, somatic embryogenesis, napthalene-3-acetic acid, liquid medium

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 255-259

 

Micropropagation of a rare mangrove Bruguiera cylindrica L. towards conservation

Varsha Vartak1* and Mahesh Shindikar2

1Department of Botany, University of Pune, Pune 411 007, India

2Geology and Palaeontology Group, Agharkar Research Institute, Pune 411 004, India

Received 20 July 2006; revised 10 May 2007; accepted 28 September 2007

An in vitro propagation protocol has been developed for Bruguiera cylindrica L. (Rhizophoraceae), a rare tree mangrove found along the western coast of India. Hypocotyl segments of viviparous propagule were cultured in vitro on Murashige and Skoog’s medium (MS). Shoot and root initiation was better on modified MS (MSM) than on MS. Shoots and roots were produced from the cut surface simultaneously in 74% explants on MSM devoid of growth regulators. The explants produced several visible shoot buds on their cut surfaces, though only 1-3 shoots were developed per explant. Multiple shoots were produced by 70% explants. When cultures were initiated in monsoon, 1.6-fold increase in shoot formation and 2.4-fold increase in root formation was observed over those initiated before monsoon. During acclimatization the survival was 87%. Considering the status of this species in terms of its distribution, threats and conservation values in the study area, its in vitro propagation will compliment and strengthen the large-scale plantation activities towards the conservation or restoration of mangrove forests.

Keywords: Bruguiera cylindrica L., conservation, hypocotyl, in vitro propagation, tree mangrove

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 260-265

 

Rapid in vitro multiplication of Drosera burmanii Vahl.: A vulnerable and medicinally important insectivorous plant

K Jayaram and M N V Prasad*

School of Life Sciences, Department of Plant Sciences, University of Hyderabad, Hyderabad 500 046, India

Received 16 October 2006; revised 30 May 2007; accepted 20 August 2007

A protocol has been stardardised for the rapid and large-scale in vitro multiplication of the vulnerable medicinal herb, Drosera burmanii Vahl. by enhanced axillary bud proliferation from shoot tip  explants. In order to standardize in vitro multiplication, the effects of different strengths of Murashige and Skoog (MS) medium (1/4, 1/3, 1/2, and full strength), different percentages of sucrose (1%, 2% and 3%), various pH (3.7, 4.7, 5.7 and 6.7) and MS basal medium fortified with different concentrations of kinetin (Kn) and 6-benzylaminopurine (BAP) (0.1, 0.5, 1.0 and 2.0 mg L-1 ) were tried on shoot tip explants.  Maximum number of multiple shoots developed on MS medium supplemented with Kn (1.0 and 2.0 mg L-1) and BAP (0.5, 1.0 and 2.0 mg L-1) separately. Direct plantlet regeneration from the leaves and in vitro flowering were also observed. Rooting was best achieved on MS basal medium. This protocol could be useful for large-scale production of biomass for quercetin, plumbagin bioprospection and long term in vitro conservation.

Keywords: Drosera burmanii, in vitro multiplication, plumbagin, quercetin, shoot tips

 

Indian Journal of Biotechnology

Vol 7, April 2008, pp 266-269

 

In vitro studies on development of gametophyte, sex-ontogeny and reproductive biology of a threatened fern, Microsorium punctatum (L.) Copel.

Ruchi Srivastava, Jyoti Srivastava, Sandip K Behera and P B Khare*

Pteridology Laboratory, National Botanical Research Institute, Lucknow 226 001, India

Received 18 November 2006; revised 9 August 2007; accepted 15 October 2007

Studies on reproductive biology vis-a-vis conservation of threatened state of the species, Microsorium punctatum, is the major concern of the present study. The life cycle starting from spore germination, gametophyte growth and differentiation, sex ontogeny and success of sporophyte formation through intra-and inter-gametophytic selfing was scrutenised and reproductive biology of this species was completely studied. The result indicates that the species could moderately be a good colonizer, as considerable number of sporophytes was produced through selfing, but in contrast very few plants were observed in the area of its occurrence. The threatened state could be due to unfavourable conditions for gametophyte growth, mating, subsequent slow pace of sporophyte development, and both epiphytic and terrestrial habit of the species.

Keywords: in vitro, reproductive biology, threatened, homosporous fern, Microsorium punctatum

Indian Journal of Biotechnology

Vol 7, April 2008, pp 270-272

 

Short Communication

 

Enhanced accumulation of triterpenoids and flavonoids in cell suspension cultures of Azadirachta indica (A. Juss.) with an extended stationary phase

Vidhu Sankar Babu1, Narasimhan S and G M Nair*

Department of Botany, University of Kerala,
Kariavattom 695 581, India

Received 7 December 2006; revised 17 August 2007;
accepted 28 October 2007

Accumulation of three triterpenoids (azadirachtin-A, nimbin and salannin) and two flavonoids (quercetin and kaempferol) was analyzed in cell suspension cultures of Azadirachta indica A. Juss. By feeding cells with a sterile solution of sucrose, it was possible to sustain viability of cells in batch cultures without subculturing for 60 d. Such long-term cell suspension cultures exhibited an extended steady stationary phase which facilitated enhanced accumulation of azadirachtin-A, nimbin, salannin, quercetin and kaempferol. 40% of the cells were viable towards the end of 60 d of incubation. Maintaining cells for longer time under stationary phase facilitated transformation of cells from low productivity to high productivity. The cells in stationary phase could serve as a starting material for the selection of elite cell lines and for bioreactor cultures for large-scale production of these compounds.

Keywords: Azadirachta indica, azadirachtin, kaempferol, nimbin, quercetin, salannin, stationary phase, suspension culture