Indian Journal of Biotechnology

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VOLUME 5 CODEN:IJBNAR 5(2) 129-258 (2006)

NUMBER 2

APRIL 2006 ISSN:0972-5849

 

CONTENTS

 

Review
Microbiological and biotechnological aspects of biodegradable plastics: Poly(hydroxyalkanoates)	137
IPC Code: Int. Cl.8 C08G63/664; C12R1:05, 1:065, 1:19, 1:38, 1:40
Nehal Thakor, Ujjval Trivedi & K C Patel
Papers
Polygalacturonases: Active site analyses and mechanism of action	148
IPC Code: Int. Cl.8 C12N9/38, 9/40
P Palanivelu
Purification and immobilization of an Aspergillus terreus xylanase: Use of continuous fluidized bed column reactor	163
IPC Code: Int. Cl.8 A01N63/02; C12N9/24; C12N11/04
Anju Pal, Lalitagauri Ray & Parimal Chattopadhyay
Bioremediation of pendimethalin contaminated soil by augmented bioslurry phase reactor operated in sequential batch (SBR) mode: Effect of substrate concentration	169
IPC Code: Int. Cl.8 C08K5/18
P N Sarma, S Venkata Mohan, M Rama Krishna & S Shailaja
Alcoholysis of vegetable oil catalyzed by an isozyme of Candida rugosa lipase for production of fatty acid esters	175
IPC Code: Int. Cl.8 C12N9/20; C12R1:72
Ahindra Nag
Production of extracellular lipase by Bacillus megaterium AKG-1 in submerged fermentation	179
IPC Code: Int. Cl.8 C12N9/20; C12R1:11
Anurag Sekhon, Neetu Dahiya, Ram P Tewari & Gurinder S Hoondal
b-Galactosidase from Lactobacillus acidophilus isolated from fermented ragi (Eleusine coracana)	184
IPC Code: Int. Cl.8 C12N19/38; C12R1:23
S K Akolkar, A D Sajgure & S S Lele
Growth hormone gene polymorphism in Kadaknath breed of poultry	189
IPC Code: Int. Cl.8 C12N15/10, 15/16
M S Thakur, S N S Parmar, T C Tolenkhomba, P N Srivastava, C G Joshi, D N Rank, J V Solanki & P V A Pillai
Cytochrome oxidase I sequence of Helicoverpa (Noctuidae: Lepidoptera) species in India—Its utility as a molecular tool	195
IPC Code: Int. Cl.8 C12N15/10, 15/52
S Kranthi, K R Kranthi, A A Bharose, S N Syed, C S Dhawad, R M Wadaskar, G T Behere & E K Patil
Distinguishing Indian commercial wheat varieties using RAPD based DNA fingerprints	200
IPC Code: Int. Cl.8 C12N15/10
G Thomas, T Mohapatra, A R Rao & R P Sharma
Alterations in RAPD profiles of proliferating shootlets of sugarcane in response to thidiazuron	207
IPC Code: Int. Cl.8 A01H4/00; C12N15/10
A K Dhawan, Rita Moudgil, Jaipal Singh Dendsay & R P Mandhan
Production and characterization of arabinogalactan protein (AGP) from a hairy root line of Catharanthus roseus (L.) G. Don	211
IPC Code: Int. Cl.8 A01H4/00; A61K35/78; C12N15/10
Nandita Mishra, Sanjay Pal, T K Maiti & Satyahari Dey
Somatic embryo proliferation in Commiphora wightii and evidence for guggulsterone production in culture	217
IPC Code: Int. Cl.8 A01H4/00; A61P3/06
Sandeep Kumar, Meeta Mathur, A K Jain & K G Ramawat
Effect of different factors on non-symbiotic seed germination, formation of protocorm-like bodies and plantlet morphology of Cleisostoma racemiferum (Lindl.) Garay	223
IPC Code: Int. Cl.8 A01H4/00
Temjensangba & Chitta Ranjan Deb
Short Communications
Development of DNA probe for detection of egg drop syndrome-76 virus	229
IPC Code: Int. Cl.8 C12N15/10
K L Kavitha, V D P Rao & V V S Suryanarayana
Development of RT-PCR based method for detection of potato virus Y in tobacco and potato	232
IPC Code: Int. Cl.8 C12N15/10
S B Ghosh & V A Bapat
Vitrification of water buffalo (Bubalus bubalis) fetal skin fibroblast cells	236
IPC Code: Int. Cl.8 A23B4/09
C R Meena & S K Das
Rapid and recurrent in vitro mass-multiplication of androgenic rice embryos	239
IPC Code: Int. Cl.8 A01H4/00
Bidhan Roy & Asit B Mandal
Somatic embryogenesis and plant regeneration in Soybean [Glycine max (L.) Merr.]	243
IPC Code: Int. Cl.8 A01H4/00
B D Ranjitha Kumari, A Settu & G Sujatha
In vitro propagation of Justicia gendarussa Burm. f.–A medicinal plant	246
IPC Code: Int. Cl.8 A01H4/00
P Agastian, Lincy Williams & S Ignacimuthu
A protocol for in vitro regeneration of Eryngium foetidum L.	249
IPC Code: Int. Cl.8 A01H4/00
M C Gayatri, M Madhu, R Kavyashree  & S P Dhananjaya
Mangrove extracts prevent the blood coagulate!	252
IPC Code: Int. Cl.8 C12P19/04
K Kathiresan, Vinoth S Ravindran & A Muruganantham
Instructions to Contributors	255

 

 

 

AUTHOR INDEX

 

 

 

 

Indian Journal of Biotechnology 

Vol 5, April 2006, pp 137-147

 

 

Microbiological and biotechnological aspects of biodegradable plastics: Poly(hydroxyalkanoates)

 

Nehal Thakor, Ujjval Trivedi and K C Patel*

Poly(3-hydroxyalkanoates) (PHAs) have been drawing much attention as biodegradable substitutes for conventional non-biodegradable plastics. Intensive research on the physiology, biochemistry and molecular genetics of the metabolism of PHAs during last two decades has increased our knowledge on the biosynthesis of these polyesters in bacteria and also showed various applications of these polymers. Prokaryotes synthesize a wide range of different PHAs and accumulate them as insoluble inclusions in the cytoplasm for storage of carbon and energy. Naturally, PHAs are synthesized from coenzyme A thioesters of the hydroxyalkanoic acids, which are synthesized during fatty acid metabolism. Due to similarities of physical and material properties with conventional plastics, PHAs can be recommended for application in various areas like industries, medicine, pharmacy and agriculture. They are thermoplastic and/or elastomeric, insoluble in water, enantiomerically pure, non toxic, biocompatible, piezoelectric and exhibit a high degree of polymerization. For economical production of PHAs, various bacterial strains have been exploited with new fermentation strategies and cheap renewable carbon sources. Transgenic plants have been studied for production of PHAs to compete with production cost of petroleum based bulk plastics. Metabolic engineering approaches have been used to expand the spectrum of utilizable substrate and also to improve PHAs production.

Keywords: biodegradable plastics, biodegradation, bio polyesters, PHAs, PHB

IPC Code: Int. Cl.⁸ C08G63/664; C12R1:05, 1:065, 1:19, 1:38, 1:40

 

Indian Journal of Biotechnology

Vol 5, April 2006, pp 148-162

 

 

Polygalacturonases: Active site analyses and mechanism of action

P Palanivelu*

Polygalacturonases from various sources have been analyzed by ClustalW and T-COFFEE, for identification of conserved and functional motifs in them. All the 104 polygalacturonases analyzed by the above programs, revealed four highly conserved motifs, viz., NTD, G/QDD, G/SHG and RIK among them. Distance conservation between the motifs was also observed. Based on the available evidences from chemical modification studies on active site amino acids, site-directed mutagenesis, protein sequence analysis and X-ray crystallographic data, a mechanism of action is proposed for this group of enzymes.

Keywords:     polygalacturonases, protein sequence analysis, active site amino acids, distance conservation between motifs, mechanism of action

 

Indian Journal of Biotechnology

Vol 5, April 2006, pp 163-168

 

 

Purification and immobilization of an Aspergillus terreus xylanase:
Use of continuous fluidized bed column reactor

 

Anju Pal, Lalitagauri Ray and Parimal Chattopadhyay*

An Aspergillus terreus extracellular xylanase produced by solid state fermentation was purified and characterized. A 6.4 fold purified xylanase was obtained by ammonium sulphate (in 30-40% saturation) precipitation followed by dialysis. Molecular weight of the xylanase was 67.0 kDa as estimated by SDS-PAGE. The enzyme was immobilized in barium alginate gel. Both free and immobilized enzyme showed maximum activity at pH 5.5 and 60°C (8.0 IU/mg & 5.25 IU/mg, respectively) and were most stable at pH 5.5 and thermostable up to 55°C. Co²⁺ stimulated free enzyme activity (9.27 IU/mg) and Mg²⁺ stimulated activity of immobilized enzyme (5.54 IU/mg). V_max and K_m for free and immobilized xylanases were 6.6 mmole/mg/min, 0.75% and 1.25 mmole/mg/min, 0.625%, respectively. 23.4% conversion of substrate (0.1% birchwood xylan) was possible in 7 h using a continuous fluidized bed column reactor under the following conditions: bed height 16 cm, temperature 40°C, and dilution rate 4.09 h^-1.

Keywords: xylanase, purification, immobilization, continuous conversion

IPC code: Int. Cl.⁸ A01N63/02, C12N9/24, C12N11/04

IPC Code: Int. Cl.⁸ C12N9/38, 9/40

Indian Journal of Biotechnology

Vol 5, April 2006, pp 169-174

 

 

Bioremediation of pendimethalin contaminated soil by augmented bioslurry
phase reactor operated in sequential batch (SBR) mode:
Effect of substrate concentration^†

 

P N Sarma*, S Venkata Mohan, M Rama Krishna and S Shailaja

Bioslurry phase reactor augmented with ETP microflora was studied for the degradation of pendimethalin contaminated soil. The effect of substrate concentration on the process performance was evaluated in the slurry phase reactor operated in sequence batch mode under anoxic-aerobic-anoxic microenvironment with a total cycle period of 120 h. The performance of augmented slurry phase system was found to be effective among the studied experiments. The degradation of the substrate in slurry system was found to be governed by the substrate loading rate. The system performance was sustained upto 5000 mg/g of substrate loading. Increase of concentration above 7,500 mg/g resulted in process inhibition. The degradation of pendimethalin in slurry phase system was found to be compared to the degradation in the soil phase. The half-life of pendimethalin in slurry phase system was found to be about 40 h which was quite rapid compared to the rate of degradation in the soil phase. The augmented slurry phase system coupled with sequential batch mode operation appears to have resulted in effective performance at higher substrate concentration.

Keywords: pendimethalin, slurry phase reactor, soil remediation, sequential batch reactor, bioaugmentation, half-life period

IPC Code: Int. C.⁸ C08K5/18

Indian Journal of Biotechnology

Vol 5, April 2006, pp 175-178

 

 

Alcoholysis of vegetable oil catalyzed by an isozyme of Candida rugosa lipase for production of fatty acid esters

Ahindra Nag*

Celite immobilized Lipase 4, an isoenzyme of commercial Candida rugosa lipase (CRL) obtained from the fungus, Candida rugosa has been catalyzed alcoholysis in dry ethanol of various triglycerides and soybean oil. The advantages of this process are that separation of the product is very easy and no additional solvent is required as alcohol acts both as a reactant and medium. The yield of monoglycerides affected by temperatures and different organic solvents has been discussed. The efficiency of catalyzed alcoholysis and operation stability of Lipase 4 was found to be better than CRL.

Keywords: alcoholysis, vegetable oil, catalysis, isozyme, lipase, fatty acid esters Candida rugosa

IPC Code: Int. Cl.⁸ C12N9/20; C12R1:72

Indian Journal of Biotechnology

Vol 5, April 2006, pp 179-183

 

 

 

Production of extracellular lipase by Bacillus megaterium AKG-1 in submerged fermentation

Anurag Sekhon¹, Neetu Dahiya², Ram P Tewari³ and Gurinder S Hoondal^3*

Bacillus megaterium AKG-1, a soil isolate, has been found to produce thermostable lipase during submerged fermentation (SF). Mannitol at a concentration of 0.2% (w/v) was the best carbon source for lipase production (848 units/mL). Among oils, soyabean oil gave highest lipase yield (1160 units/mL), followed by coconut oil (912 units/mL). Lipase yield was maximum (1000 units/mL) with wheat bran (1%) as sole carbon and nitrogen source, followed by neem seed cake (810 units/mL) and cotton seed cake (790 units/mL).

Keywords: B. megaterium AKG-1, submerged fermentation, optimization, carbon source

IPC Code: Int. Cl.⁸ C12N9/20; C12R1:11

Indian Journal of Biotechnology

Vol 5, April 2006, pp 184-188

 

 

b-Galactosidase from Lactobacillus acidophilus isolated from fermented ragi (Eleusine coracana)

S K Akolkar, A D Sajgure and S S Lele*

Lactobacillus acidophilus was isolated from fermented millet, Eleusine coracana. It was characterized using several biochemical tests. This strain was found to be homofermentative, slime forming and b-galactosidase producer. Recovery and characterization of b-galactosidase were studied at laboratory scale. Since the enzyme was intracellular various cell lysis methods were studied to achieve maximum enzyme release from cell fragments. Homogenization at a pressure of 2000 psig two passes showed an enzyme release of 2050 U/mL and was found the best method for cell lysis. The cell extract was purified using ultrafiltration and gel permeation chromatography. Specific activity and fold purification of b-galactosidase was found to be 568.61 and 21.2, respectively. Kinetic parameters were determined using ONPG (o-nitrophenyl galactopyranoside) as a substrate. Michaeli’s Menten equation was found to fit the reaction of ONPG using b-galactosidase from L. acidophilus. Vmax and Km were calculated from the Lineweaver Burk plot. Vmax was found to be 4.94 min^-1 and Km to be 0.11 mM. Using gel permeation chromatography, the molecular weight was found to be in the range of 450-500 kDa.

Keywords: Lactobacillus acidophilus, Eleusine coracana, b-galactosidase

IPC Code: Int. Cl.⁸ C12N 19/38; C12R1:23

Indian Journal of Biotechnology

Vol 5, April 2006, pp 189-194

 

 

Growth hormone gene polymorphism in Kadaknath breed of poultry

 

M S Thakur¹, S N S Parmar¹*, T C Tolenkhomba¹, P N Srivastava¹, C G Joshi², D N Rank²,
J V Solanki² and P V A Pillai¹

 

The chicken growth hormone gene of intron I region from three varieties of Kadaknath breed of poultry was amplified by PCR and a 770 bp product was obtained. The products from each variety (Jet black, Golden and Pencilled) were digested with Msp I (5 U), which recognizes the 5¢-C ¯ C G G-3¢ sequence. The RFLP pattern revealed restriction fragments of 529, 373, 241 and 156 bp sizes, which indicated the presence of two restriction sites. In total, three RFLP patterns was observed at two restriction sites. The genotypic frequencies obtained for different varieties were tested for equilibrium using chi-square test. The differences among genotypes for all the three varieties were found to be non-significant, which indicated that population was in Hardy-Weinberg equilibrium. The alleles A and A₂ were not observed in all the three varieties of Kadaknath breed of poultry. The overall allelic frequencies for A₁ and A₃ alleles were 0.375±0.016 and 0.625±0.020, respectively for all the three varieties. The highest allelic frequency (0.700±0.011) was obtained for A₃ allele in Pencilled variety and lowest (0.300±0.022) for A₁ allele was also observed in Pencilled variety. The phylogenetic consensus tree grouped all the three varieties into one cluster. The highest genetic distance (0.0748) was observed between Jet black and Pencilled and smallest (0.0051) between Jet black and Golden varieties. The genetic differences among all the three varieties based on phylogenetic tree were negligible; which indicates non-significant differences among each other. Thus it can be concluded that all the three varieties belonging to the Kadaknath breed of poultry have almost similar genetic base.

Keywords: cGH gene polymorphism, genetic distance, indigenous chicken, phylogenetic

IPC Code: Int. Cl.⁸ C12N15/10, 15/16

Indian Journal of Biotechnology

Vol 5, April 2006, pp 195-199

 

 

Cytochrome oxidase I sequence of Helicoverpa (Noctuidae: Lepidoptera) species in India—Its utility as a molecular tool

S Kranthi, K R Kranthi, A A Bharose, S N Syed, C S Dhawad, R M Wadaskar, G T Behere and E K Patil

The genus Helicoverpa comprises of 2 species in India—H. armigera and H. assulta. This paper compares partial CO-1 sequences of the two-field-collected species with a laboratory strain of H. armigera and Drosophila yakuba whose entire mitochondrial genome has been sequenced. The region sequenced in the study corresponds to 2111 to 2601 bp sequence of D. yakuba mitochondrial genome, i.e mid to near terminal segment of the CO-I region. When analyzed, at least 18 nucleotide and 8 amino acid substitutions were observed between the two species. Using this information, a specific PCR-RFLP tool was designed that distinguishes between the two species at the egg stage itself, thus, influencing pest control options significantly, especially in areas and crops where the two may occur simultaneously.

Keywords: CO-I sequence, Drosophila yakuba, Helicoverpa assulta, H. armigera

IPC Code: Int. Cl.⁸ C12N15/10, 15/52

Indian Journal of Biotechnology

Vol 5, April 2006, pp 200-206

 

 

Distinguishing Indian commercial wheat varieties using
RAPD based DNA fingerprints

 

G Thomas², T Mohapatra¹, A R Rao³ and R P Sharma^1*

Wheat is a major contributor to food self-sufficiency in India. A large number of varieties have been bred over years to sustain higher production levels. These varieties have been described primarily by morphological characteristics, which show stage and tissue specific expression, and interaction with environment. The present study was undertaken to test the efficiency of RAPD in distinguishing and determining the extent of genetic relatedness among 96 varieties belonging to Triticum aestivum, T. durum, T. dicoccum and Triticale, using 50 random decamer primers. Eighty-two per cent of these primers and 78.8% of the amplified fragments were found to be polymorphic. The number of polymorphic fragments per primer ranged from 1-13 with an average of 4.92. The varieties could be unambiguously distinguished from each other by RAPD profiles based on all the 50 as well as 10 most informative primers, which detected more than 5 polymorphic fragments each. The estimated probability of identical match by chance suggested that the RAPD based DNA fingerprints can be used with high degree of confidence for establishing distinctness of the varieties. Average similarity (75%) among the hexaploids was the same as in the tetraploids, although the range was broader in case of the former group of varieties. This suggested a relatively narrow genetic base of the tetraploid varieties as compared to the hexaploids. Cluster and principal component analysis clearly distinguished the tetraploids from the hexaploids.

Keywords: commercial varieties, DNA fingerprinting, RAPD, Triticale, Triticum aestivum, T. dicoccum, T. durum.

IPC Code: Int. Cl.⁸ C12N15/10

Indian Journal of Biotechnology

Vol 5, April 2006, pp 207-210

 

 

Alterations in RAPD profiles of proliferating shootlets of sugarcane in response to thidiazuron

A K Dhawan¹*, Rita Moudgil², Jaipal Singh Dendsay¹ and R P Mandhan²

Proliferating shootlets of sugarcane varieties CoH 92 and Co 7717, raised from apical meristems, were cultured in vitro for 24 d in MS medium with different concentration of TDZ. DNA from leaves of these shootlets was extracted and amplified using 10 random decamer primers. A total of 50 RAPD products in var. CoH 92 and 43 in var. Co 7717 were formed. TDZ caused considerable differences in amplification products of both varieties. Several RAPD products, which were absent in control plantlets, amplified in TDZ treatments, such as 950 bp (CoH 92 and Co 7717) against primer 5´-AATCGGGCTG-3´ (10A04); 1200 bp (Co 7717) against primer 5´-AGCCAGCGAA-3´ (50A16); 1150 and 1000 bp (CoH 92 and Co 7717) and 900 bp (Co 7717) against primer 5´-CTGCTGGGAC-3´ (80B10); 950 bp (CoH-92) and 500 bp (Co 7717) against primer 5´-GTAGACCCGT-3´ (90B11); and 500 bp (CoH 92) against primer 5´-CCGCATCTAC-3´ (100C04). On the other hand, certain RAPD products, such as 220 bp (CoH 92) against primer 5´-TCGGCGATAG-3´ (40A10); 950 bp (Co 7717) against primer 50A16; 550, 360 and 320 bp (CoH 92) against primer 5´-GGACTGGAGT-3´ (60B04); and 1050 bp (CoH 92) against primer 80B10, amplified in the control plantlets disappeared due to TDZ treatments. Such altered RAPD profile in response to TDZ is an entirely new and hitherto unsuspected observation, which suggests a possible genetic change or perhaps to a synthesis of protein or non-protein regulators that affect the process of DNA synthesis/amplification.

Keywords: RAPD, shoot multiplication, sugarcane, thidiazuron

IPC Code: Int. Cl.⁸ A01H4/100; C12N15/10

Indian Journal of Biotechnology

Vol 5, April 2006, pp 211-216

 

 

Production and characterization of arabinogalactan protein (AGP) from a hairy root line of Catharanthus roseus (L.) G. Don

 

Nandita Mishra, Sanjay Pal, T K Maiti and Satyahari Dey*

Arabinogalactan protein (AGP), a class of cell wall proteoglycan, was isolated from the hairy root cultures of a newly developed hairy root line IIT-BT/D1 of Catharanthus roseus (L.) G. Don. AGP was found to be present both in the roots (0.3 mg/g fresh weight) and in the spent media (47 mg/L). The compositional analysis revealed the predominance of arabinose and galactose sugar, a characteristic feature of AGP.

Keywords: Catharanthus roseus, Agrobacterium rhizogenes, hairy root culture, arabinogalactan protein (AGP)

IPC Code: Int. Cl.⁸ A01H4/00; A61K/35/78; C12N15/10

Indian Journal of Biotechnology

Vol. 54, January April 20065, pp 217-222

 

 

Somatic embryo proliferation in Commiphora wightii and evidence forguggulsterone production in culture

Sandeep Kumar¹, Meeta Mathur, A K Jain and K G Ramawat*

A method for obtaining somatic embryos consistently in Commiphora wightii (Arnott.) Bhandari has been developed. Somatic embryos when sub-cultured on modified MS medium containing ABA (10 µg L^-1) produced more embryos directly by meristematic activity of the epidermal and subepidermal cells at the hypocotyl region or also from the root tip region. Embryo proliferation was also observed on auxin-cytokinin free modified MS medium supplemented with different concentrations of activated charcoal, abscisic acid, mannitol and sucrose. Modified MS hormone-free medium containing activated charcoal 0.5 g L^-1 and sucrose 10 g L^-1 was most suitable for multiplication and maturation of embryos. Embryo formation was also recorded from callus produced by earlier formed somatic embryos (indirect). Embryos obtained from various maturation treatments when desiccated under laminar airflow bench for 5-9 d showed maximum germination percentage. Guggulsterone-E and -Z contents of in vitro embryonic cultures were ¼ - and ½ -fold of that found in zygotic embryos, but several fold higher than callus cultures, therefore, hold promise for the production of bioactive guggulsterones.

Keywords: Commiphora wightii, somatic embryogenesis, guggulsterones, antihyperlipidemia, callus cultures

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

Indian Journal of Biotechnology

Vol 5, April 2006, pp 223-228

 

 

Effect of different factors on non-symbiotic seed germination,
formation of protocorm-like bodies and plantlet morphology of
Cleisostoma racemiferum (Lindl.) Garay

 

Temjensangba and Chitta Ranjan Deb*

Cleisostoma racemiferum, an epiphytic orchid of primary forest under threat in their natural habitat, was studied. Immature seeds of different developmental stages [(8-20 week after pollination (WAP)] were cultured on Knudson ‘C’, Mitra et al and MS media supplemented with sucrose (0-3%, w/v), coconut water (CW, 0-20%, v/v) and NAA (0-30 mM) + BA (0-8 mM) singly or in combination. After 7 week of culture, first sign of germination was recorded as nodular swelling of seeds. Amongst the three different basal media tested, better germination was supported by MS medium, followed by Mitra et al and Knudson ‘C’ media containing sucrose (3%) and NAA (10.0 mM ) + BA (8.0 mM). Of various developmental stages of the seed, better germination was obtained from green pods of 16 week after pollination. Younger seeds did not show any sign of germination, while mature seeds exhibited delayed and deformed germination. Though incorporation of CW in the medium did not show much influence on seed germination, but 15% (v/v) CW in the initiation medium enhanced the early differentiation of protocorm-like bodies (PLBs) into plantlets. Within 14-16 week of culture on germination medium, the PLBs started releasing the first set of leaflets. The advanced stage PLBs were converted into rooted plantlets on MS medium containing IAA (10.0 mM) + Kn (9.0 mM). Although the medium containing NAA (10.0 mM) + BA (8.0 mM) resulted in multiple shoot buds, but the leaves were thin and etiolated. Further, the medium enriched with NAA (10.0 mM) + Kn (9.0 mM) resulted in stunted growth of plantlets, while presence of IAA (10.0 mM) + BA (8.0 mM) resulted in plantlets with poor roots.

Keywords: Cleisostoma racemiferum, green pod culture, plant growth regulators, seed age

IPC Code: Int. Cl.⁸ A01H4/00

Short Communications

Indian Journal of Biotechnology

Vol. 5, April 2006, pp.229-231

 

 

Development of DNA probe for detection of egg drop syndrome-76 virus

K L Kavitha*, V D P Rao¹ and V V S Suryanarayana²

Two kb, BamHI fragment of duck embryo propagated Indian isolate (VN 1) of egg drop syndrome-76 (EDS-76) was cloned into pBluescript II KS + vector and the fragment was labelled with a ³³p dATP label using random primer extension method. The labelled DNA was used as a probe for detecting viral DNA in faecal samples of experimentally infected birds. Proteinase K treated, phenol extracted, alkali denatured samples exhibited strong signals as compared to proteinase K digested samples alone. The probe could detect up to 1.25 pg of viral DNA. However, it did not show any positive signal with another adenovirus, i.e., hydropericardium syndrome viral DNA indicating its specificity for egg drop syndrome virus (EDSV).

Keywords: Egg drop syndrome-76, Indian isolate, DNA probe, radiolabelling, specificity, sensitivity

IPC Code: Int. Cl.⁸  C12N15/10

Indian Journal of Biotechnology

Vol. 5, April 2006, pp.232-235

 

 

Development of RT-PCR based method for detection of potato virus Y in tobacco and potato

S B Ghosh and V A Bapat*

A reverse transcription polymerase chain reaction (RT-PCR) based protocol with a pair of potato virus Y coat protein gene specific primers was found to be more sensitive method for the PVY detection in infected tobacco leaf and potato tuber tissues than conventional enzyme linked immunosorbent assay (ELISA) or nucleic acid spot hybridization (NASH) based methods. It was found in our studies that by RT-PCR, the presence of the virus could be detected in potato tubers of two field samples, which could not be detected by NASH or ELISA.

Keywords: ELISA, NASH, potato virus Y (PVY), RT-PCR

IPC Code:  Int. Cl.⁸  C12N15/10

Indian Journal of Biotechnology

Vol 5, April 2006, pp 236-238

 

 

Vitrification of water buffalo (Bubalus bubalis) fetal skin fibroblast cells

C R Meena and S K Das¹*

A method was developed for the cryopreservation of buffalo fetal skin fibroblast cells by vitrification. Skin cells were isolated from 1-2-month-old fetuses obtained from an abattoir, by enzymatic digestion (0.5% w/v trypsin + 0.05% w/v collagenase in DPBS) for 15-20 min. They were washed 5-6 times with DPBS and then once with RPMI-1640 + 10% FBS by centrifugation at 600 rpm. The cells were then cultured in the same medium in a CO₂ incubator (5% CO₂ in air) at 38.5°C for 3 d. After 3 days, the fibroblast cells from the monolayer were harvested by treating with trypsin-versene solution. They were equilibrated and loaded into 0.25 mL French straw, containing 0.5 M sucrose solution in DPBS. The straws were sealed and precooled by keeping them in liquid nitrogen (LN₂) vapour for 1-2 min, following which these were plunged into LN₂. After 5 days of storage the straws were warmed quickly by transferring them to a water bath at about 20ºC for 15-20 sec. The cells were collected by centrifugation and plated in 25 cm² culture flasks with 5-6 mL of culture media and incubated at 38.5º±1ºC with 5% CO₂ in humidified (95%) incubator. The fibroblast cells growth started within 3 h of culture set-up and on day 3 the monolayer formation occurred.

Keywords: buffalo, cryopreservation, fetal skin fibroblasts, vitrification

IPC Code: Int. Cl.⁸ A23B4/09

Indian Journal of Biotechnology

Vol 5, April 2006, pp 239-242

 

 

Rapid and recurrent in vitromass-multiplication of androgenic rice embryos

 

Bidhan Roy and Asit B Mandal*

In vitro mass multiplication of plantlets of androgenic origin was achieved in an indica rice var. IR72. The multiple shoots were developed profusely on MS medium containing 4 mg L^-1 Kn. When the shoots were cultured on MS supplemented with 6 mg L^-1 BAP, a large number of dormant embryos, pro-embryos and embryo-like structures were produced. Those multiple shoots and dormant embryos had no roots. The subcultured shoots and the dormant embryos on MS basal developed into healthy plantlets with prolific roots. Since 1998, these dormant embryos and the shoots are being sub-cultured at 45 d interval. Till date the multiple shoots and the dormant embryos are developing constantly depending upon the hormones used in the medium. The stock is being maintained by recurrent subculture using explants from the preceding culture. Such stock offers ample scope in genetic transformation of indica rice and multiplication of transgenic plantlets. The protocol also prospects efficient in vitro conservation through repeated subculture. It provides a novel source too for constant supply of embryos to produce synthetic seeds of androgenic origin in rice.

Keywords: anther culture, microtillering, Oryza sativa, IR72

IPC Code: Int. Cl.⁸ A01H4/00

Indian Journal of Biotechnology

Vol 5 April 2006, pp 243-245

 

 

Somatic embryogenesis and plantregeneration in Soybean [Glycine max (L.) Merr.]

B D Ranjitha Kumari*, A Settu and G Sujatha

Establishment of efficient protocol for the induction of somatic embryogenesis and plant regeneration from embryonic axes explants was achieved in Glycine max (L.) Merr. Highest frequency (92.9%) of embryogenic callus formation was obtained from embryonic axes explants of cv. CO-1 on MS media containing 2,4-Dichlorophenoxy acetic acid (2,4-D) and 6-benzyl adenine (BA). Fast growing, yellowish nodular callus lines containing somatic embryos were established on initiation medium supplemented with 180.8 µM 2,4-D and 2.22 µM BA, respectively. Embryo development and maturation was achieved on MS medium supplemented with 2.26 µM 2,4-D and 8.88 µM BA. The well formed embryos germinated into complete plantlets on MS medium containing 8.88 µM BA and 1.34 µM NAA (a-Naphthalene acetic acid). The regenerated plants were first transferred to plastic cups and later into pots for maturation. Regenerated plants did not exhibit observable morphological alterations.

Keywords: embryonic axes, callus, somatic embryogenesis, plant regeneration, Glycine max

IPC Code: Int. Cl.⁸ A01H4/00

Indian Journal of Biotechnology

Vol 5, April 2006, pp 246-248

 

 

In vitro propagation of Justicia gendarussa Burm. f.–A medicinal plant

P Agastian, Lincy Williams and S Ignacimuthu^·

An efficient protocol for in vitro propagation of Justicia gendarussa Burm. f. has been developed. MS medium supplemented with NAA induced prolific callus in both leaf and nodal explants. Organogenic and chlorophyllous calli were produced at lower concentrations of NAA (1.0 mg L^‑1) and BAP (0.1 mg L^-1). Thick and long roots with numerous root hairs were produced with NAA (1.0 mg L^-1) and BAP (0.1 mg L^-1). Long shoots were also formed. Of the in vitro grown 120 plantlets transferred to the field 94% survived after 2 months of transplantation to natural environment.

Keywords: in vitro propagation, organogenesis, Justicia gendarussa

IPC Code: Int. Cl.⁸ A01H4/00

Indian Journal of Biotechnology

Vol 5, April 2006, pp 249-251

 

 

A protocol for in vitro regeneration of Eryngium foetidum L.

M C Gayatri *, M Madhu, R Kavyashree and S P Dhananjaya

Eryngium foetidum L. is an important plant cultivated as leafy vegetable and for its essential oil, which is of high economical value in international market. Multiple shoots (17.8) were obtained through direct regeneration from leaf excised from field grown plants of E. foetidum and cultured on Linsmaier and Skoog’s Basal Medium (LSBM) fortified with BAP (1.5 mgl^-1) and PVP (250 mgl^-1). After subculture on the same medium, the multiple shoots formed roots, thereby eliminating an additional step of in vitro rooting. The well-developed plants were hardened and successfully transferred to field with 88% survival frequency.

Keywords:            Eryngium foetidum, leaf, direct regeneration, multiple shoots, survival frequency

IPC Code: Int. Cl.⁸ A01H4/00

Indian Journal of Biotechnology

Vol 5, April 2006, pp 252-254

 

 

 

Mangrove extracts prevent theblood coagulate!

K Kathiresan*, Vinoth S Ravindran and A Muruganantham

Mangrove polysaccharide extracts prolonged the time taken for blood clotting, and this was also more pronounced with the assay of activated partial thromboplastin time (APTT), than that of prothrombin time (PT). This activity increased with concentrations of extracts used (100, 500, and 1000 µg mL^-1) and also with the levels of sulphate present in the samples. The anticoagulant activity was high in Avicennia marina and Aegiceras corniculatum compared to Excoecaria agallocha and Rhizophora spp.

Keywords: mangroves, polysaccharides, blood anticoagulant, Avicennia marina, Aegiceras corniculatum, Rhizophora sp.

IPC Code: Int. Cl.⁸ C12P19/04