Indian Journal of Biotechnology

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VOLUME 5 CODEN:IJBNAR 5(1) 1-128 (2006)

NUMBER 1

JANUARY 2006 ISSN:0972-5849

 

CONTENTS

 

Reviews
Regulation of gene expression through post-initiation controls	9
IPC Code: Int. Cl.7 C07H21/02; C12P19/32
S Vidyalakshmi & V Ramamurthy
Biocontrol of wood-rotting fungi	20
IPC Code: Int. Cl.7 A01N63/02
Dishant Kumar & Rajinder K Gupta
Papers
Stable integration, expression and inheritance of the ferritin gene in transgenic elite indica rice cultivar BR29 with enhanced iron level in the endosperm	26
IPC Code: Int. Cl.7 C12N15/09, 15/29
M Khalekuzzaman, K Datta, N Oliva, M F Alam, 0 I Joarder & S K Datta
Isolation and characterization of gene encoding vicilin (7S) protein from cDNA clones of immature seeds of pigeon pea [Cajanus cajan (L.) Millsp.]	32
IPC Code: Int. Cl.7 C12N15/11
Subodh Gupta, A K Verma & Laxmi Chand
Development of monoclonal antibodies against group A animal rotaviruses	37
IPC Code: Int. Cl.7 C07K16/08; G01N33/53
B R Gulati, R Pandey  & B K Singh
Immune responses to inactivated oil adjuvanted Equine Herpes Virus-1 using different emulsifiers in horses	42
IPC Code: Int. Cl.7 A61K39/27
B K Singh, S N Tandon & N Virmani
Molecular characterization of different breeding groups of outbred Mongolian gerbils (Meriones unguiculatus)	47
IPC Code: Int. Cl.7 C12N15/10
B Maity & P Y Guru
Superovulation and embryo recovery in two breeds of rabbits	54
IPC Code: Int. Cl.7 A61K38/24
S Satheshkumar
Phylogentetic analysis of Mycobacterium leprae genome for identification of novel drug targets	58
IPC Code: Int. Cl.7 C12R1:32
Aditya Saxena, Pritish Varadwaj, S Singh, Manoj Jaiswal, K Misra & T Lahiri
RAPD analysis of genetic variability in Pinus gerardiana Wall. in Kinnaur (Himachal Pradesh)	62
IPC Code: Int. Cl.7 C12N15/10
Anil Kant, D Pattanayak, S K Chakrabarti, Rajan Sharma, Manisha Thakur & D R Sharma
Optimization of microbial degradation of an azo dye (methyl red) in fixed film bioreactors	68
IPC Code: Int. Cl.7 C02F3/34; C02F103:30; C12R1:645, 1:07, 1:67, 1:68, 1:685, 1:885
Suresh Kumar, K P Sharma, Shweta Sharma, Ruby Grover, Pawan Kumar, Pratima Soni & Subhasini Sharma
Production of PHB (bioplastics) using bio-effluent as substrate by Alcaligens eutrophus	76
IPC Code: Int. Cl.7 A01N63/00; C08G63/00; C12R1:05
B Senthil Kumar & G Prabakaran
Optimization of process parameters for alkaline protease production under solid-state fermentation by Thermoactinomyces thalpophilus PEE 14	80
IPC Code: Int. Cl.7 C12N9/50; C12R1:01
G Divakar, M Sunitha, P Vasu, P Udaya shanker & P Ellaiah
Immobilization of Pleurotus ostreatus 1804 on PUF cubes: Influence of mycelial growth pattern on laccase yield	84
IPC Code: Int. Cl.7 A01N63/04; C12N9/00, 11/08; C12R1:645
K Krishna Prasad, S Venkata Mohan, B R Pati & P N Sarma
Characterization of Cucumber mosaic virus infecting Indian long pepper (Piper longum L.) and betel vine (Piper betle L.) in India	89
IPC Code: Int. Cl.7 C12N7/02, 15/09; C12R1:94
P S Hareesh, R Madhubala & A I Bhat
Occurrence of Iris mild mosaic potyvirus in cultivated iris in India	94
IPC Code: Int. Cl.7 C07K16/10; C12N15/10; G01N33/53
Saurabh Kulshrestha, Vipin Hallan, Gaurav Raikhy, Raja Ram, I D Garg, Q M R Haq & A A Zaidi
Genotype dependent influence of phytohormone combination and subculturing on micropropagation of sugarcane varieties	99
IPC Code: Int. Cl.7 A01H4/00
Nandita Singh, Anil Kumar & G K Garg
Callus induction from Ipomoea aquatica Forsk. leaf and its antioxidant activity	107
IPC Code: Int. Cl.7 A01H4/00; C09K15/00
K Nagendra Prasad, M Siva Prasad, G R Shivamurthy & S M Aradhya
In vitro flowering and rapid propagation of Vitex negundo L.—A medicinal plant	112
IPC Code: Int. Cl.7 A01H4/00, 5/02
A V Vadawale, D M Barve & A M Dave
Short Communications
Amplification and cloning of canine parvovirus VP1 gene into mammalian expression vector	117
IPC Code: Int. Cl.7 C12N15/09
P K Gupta, A A Raut & A Rai
Designing specific oligonucleotide primers for metallothionein genes	120
P J Thomas, T Anand, P Suresh, S Janarthanan & S Vincent
Book Review	123
Instructions to Contributors	125

 

 

 

 

                            

                            

 

 

 

 

 

Indian Journal of Biotechnology

Vol 5, January 2006, pp 9-19

 

 

 

Regulation of gene expression through post-initiation controls

S Vidyalakshmi and V Ramamurthy*

Synthesis of messenger RNA in an eukaryotic cell can be regulated at multiple levels. Although the regulatory processes at the initiation step help in the discrimination between whether or not a gene need to be transcribed in a particular cell at a particular time, the decision to go through with the complete synthesis could still be revised through controls at the succeeding steps of the process. This article reviews the regulatory controls exercised at the transcription elongation step and the factors which participate in this process. Inspite of the plethora of factors contributing to the elongation process, the main catalytic activity is entrusted with the RNA polymerase II, and other factors mainly assist or modify the polymerase to alter its enzymatic efficiency.

Keywords: CTD phosphorylation, dephosphorylation, RNA polymerase II, transcription elongation

IPC Code:  Int. Cl.⁷ C07H21/02; C12P19/32

Indian Journal of Biotechnology
Vol. 5, January 2006, pp 20-25

 

 

 

Biocontrol of wood-rotting fungi

Dishant Kumar and Rajinder K Gupta*

Fungal decay and deterioration of softwood and hardwood trees are the most common and damaging problems of forest and timber industries worldwide. A range of microbial as well as insect deteriogens can attack wood. Although some wood types contain chemical extractives that confer resistance against wood decay fungi, most are non-durable and subject to attack by a wide range of fungi, thereby necessitating a broad spectrum controlling action. The wood preserving industry uses chemical wood preservatives that pose adverse health and environmental effects. To avoid this, new biological and biochemical control systems are needed for the preservation of wood decay. This review summarizes the state of art in research and prospective use of wood and rhizosphere-inhabiting actinomycetes as biocontrol agents for brown- and white-rot fungi.

Keywords: biocontrol agents, wood-rotting fungi, antibiosis, mycoparasitism, Streptomyces violaceusniger, chitinase

IPC Code: Int. Cl.⁷ A01N 63/02

Indian Journal of Biotechnology

Vol. 5, January 2006, pp. 26-31

 

 

 

Stable integration, expression and inheritance of the ferritin gene in transgenic elite indica rice cultivar BR29 with enhanced iron level in the endosperm

M Khalekuzzaman^1,2, K Datta^l, N Oliva^l,4, M F Alam², 0 I Joarder³ and S K Datta^l,4*

Rice (Oryza sativa L.) is a major crop providing staple diet for more than half of the world's population, but it does not fulfill the recommended daily dietary allowance. Moreover, milling of rice grain causes considerable losses of nutrients, including iron. Iron deficiency is a global nutritional problem. About 3.5 billion people in the developing world suffer from iron-deficiency anemia, of which 50% is dietary in origin. To increase iron storage in rice, we introduced the ferritin gene driven by an endosperm-specific glutelin promoter into a Bangladeshi rice cultivar, BRRl Dhan 29 (BR29), using the biolistic method. Analysis demonstrated integration, inheritance and expression of the ferritin gene up to the T₃ generation. The iron content in seeds was estimated by using the ICP (Inductively Coupled Argon Plasma) Spectrometer. All transgenic plants accumulated higher levels of iron in the grain, with as much as 9.2 mg/kg versus the control (3.8 mg/kg). A histochemical reaction of the thin microtome section revealed the presence of iron in the endosperm cells of the transgenic grain. This finding suggests that homozygous rice lines with enhanced iron content developed by genetic engineering could help overcome iron deficiency in developing countries.

Keywords: indica rice BR29, inheritance, iron accumulation, nutrition, transgenic rice

IPC code: Int. Cl.⁷ C12N15/09, 15/29

Indian Journal of Biotechnology
Vol 5, January 2006, pp 32-36

 

 

 

Isolation and characterization of gene encoding vicilin (7S) protein from cDNA clones of immature seeds of pigeon pea [Cajanus cajan (L.) Millsp.]

Subodh Gupta^*1, A K Verma and Laxmi Chand

cDNA library was constructed in lgt 11 expression vector from mRNA of immature seeds of pigeonpea.  This cDNA was screened with specific non-radioactive, DIG-labelled heterologous pea vicilin cDNA probe (pRC-758). The positive vicilin (7S) encoding cDNA clones were isolated. One of the vicilin cDNA clones showed insert size of ~1.3 Kb, when analyzed by PCR using lgt 11 forward and reverse primers. This PCR product was subcloned in pUC-18 vector for confirmation of gene and product by Southern and Western hybridizations, respectively. The clone was named as pSL-1 and sequenced with M13 universal forward and reverse sequencing primers. The partial nucleotide sequence (1341 bp) has been indexed in NCBI gene bank with accession number AF348366. The complete gene has 1417 base pairs with 972 bp coding sequence. Hence, the predicted polypeptide chain of this gene was determined, which contained 323 amino acids. After post translation modification, the predicted polypeptide has 310 amino acids long with approximately mol wt of 34.1 kDa. This gene encoding vicilin (7S) protein provides the basic understanding of the gene structure of pigeonpea storage proteins.

Key words:  cDNA, gene, pigeonpea, storage protein, vicilin (7S)

IPC Code: Int. Cl.⁷ C12N15/11

Indian Journal of Biotechnology
Vol. 5, January 2006, pp. 37-41

 

 

 

Development of monoclonal antibodies against group A animal rotaviruses

B R Gulati *, R Pandey ¹ and B K Singh

Hybridomas secreting monoclonal antibodies (MAbs) to a group A rotavirus (GAR) were developed by fusion of myeloma cell (SP2/0) with spleen cells of BALB/c mice immunized with semi-purified bovine rotavirus (CR129, G10P11). Two of these rotavirus-specific monoclonal antibodies were further characterized by immunoblotting and also tested for their reactivity with rotaviruses of bovine, equine and porcine origin in a sandwich ELISA. MAb 6C7F7 (IgM isotype) showed reactivity with two proteins i.e. VP2 (95 kDa) and VP6 (44 kDa), while MAb 2H7E8 (IgG2b isotype) reacted with only 44 kDa VP6 protein of purified bovine rotavirus CR129 on immunoblotting. These VP6-specific MAbs were used as capture antibody in sandwich ELISA for testing with different group A rotavirus antigens. Both MAbs reacted equally with all the bovine and equine rotavirus isolates tested and also with two known rotavirus positive porcine stool samples. These findings suggest that one of these MAbs is directed specifically against a group-specific protein VP6 and will be useful to detect group A rotaviruses directly from stool samples in different animal species employing a sandwich ELISA.

Keywords: rotavirus, monoclonal antibody, bovine, equine, porcine, ELISA

IPC Code: Int. Cl.⁷ C07K16/08; G01N33/53

 

Indian Journal of Biotechnology

Vol. 5, January 2006, pp. 42-46

 

 

 

Immune responses to inactivated oil adjuvanted Equine Herpes Virus-1 using different emulsifiers in horses

B K Singh^*, S N Tandon and N Virmani

Immune responses to formalin inactivated, oil adjuvanted equine herpes virus-1 (EHV-1) using Tween-80 (OET-80) and mannide monooleate (OEMM) emulsifiers were compared with a killed EHV-1 commercial vaccine in the three groups of EHV-1 seronegative Kathiawari horses. Each group of horses (n=3) was immunized with three doses each of the OET-80, OEMM and commercial vaccine. The complement fixation test (CFT) and virus neutralization test (VNT) were used to measure antibody levels in these horses. Mild complement-fixing (CF) antibody responses were observed after primary immunization with all the three products. Nearly 4-fold rise in CF antibody response after first booster in horses was seen up to 5, 6 and 9 weeks with OET-80, commercial vaccine and OEMM, respectively. Second booster CF antibody response was also seen in all the three groups. After primary immunization, VN antibody response was noticed in all the three groups. However, first booster injection of commercial vaccine, OET-80 and OEMM increased nearly 4-fold rise in VN antibody titre; which lasted up to 7, 9, and 12 weeks, respectively. VN antibody titre obtained after first booster injection of OEMM in horses was significantly higher (P≤0.05) as compared to OET-80 and commercial vaccine. After second booster injection rise in VN antibody titre was also observed in all the three groups of immunized horses. Control group of horses did not show any CF and VN antibodies responses. OEMM produced better CF and VN antibody responses than OET-80 and commercial vaccine.

Keywords: booster effect, CFT, EHV-1 immunogen, emulsifiers, horses, immunization, VNT

IPC Code: Int. Cl.⁷ A61K39/27

 

Indian Journal of Biotechnology

Vol. 5, January 2006, pp 47-53

 

 

 

Molecular characterization of different breeding groups of outbred Mongolian gerbils (Meriones unguiculatus)

B Maity* and P Y Guru

Two RAPD primers and 15 protein biochemical markers were examined to assess the zygosity (hetero/homozygocity) and per cent polymorphisms in monogamous (IM: IF) and triogamous (1M:2F) breeding groups of outbred Mongolian gerbils (Meriones unguiculatus). No polymorphism and heterozygosity were found in RAPD as well as biochemical markers in monogamous groups, whereas both primers in RAPD (%P=55%) and the seven biochemical markers (Alb-1, Es-2, Es-3, Hbb, Idh-1, Pgm-1 and Trf -1) showed polymorphism. On the basis of 15 biochemical markers, genetic variability in terms of per cent polymorphisms (%P=40%), observed heterozygosity (Hob=0.069±0.013), expected heterozygosity (He=0.214±0.277) were observed in triogamous groups. The reproductive performance was severely affected in monogamous groups (colony index, CI=0.33) due to the loss of heterozygosity within the population, whereas the best reproductive performance (CI =0.77) was achieved in heterozygous, triogamous groups.

Keywords: Mongolian gerbil (Meriones unguiculatus), breeding systems; RAPD, protein biochemical markers, cellulose acetate electrophoresis

IPC Code:   Int. Cl.⁷ C12N15/10

 

Indian Journal of Biotechnology
Vol 5, January 2006, pp. 54-57

 

 

 

Superovulation and embryo recovery in two breeds of rabbits

S Satheshkumar*

Broiler rabbit does of two breeds, viz. New Zealand White (NSW, 6) and Soviet Chinchilla (SC, 6), were subjected to superovulatory treatment by administering PMSG (150 IU; intramuscular). At the induced oestrum, each doe was allowed to mate with two bucks and HCG (150 IU; intravenous) was administered. Equal numbers of control animals from the respective breeds were allowed double mating without any hormonal treatment. In the treatment group, high intensity oestrus was recorded in NZW and SC rabbits (66.7 and 83.3%, respectively), while the control animals exhibited moderate intensity oestrus. Both, NZW and SC rabbit does responded similarly to the superovulation treatment. Thus, the breed influence could not be appreciated in the ovulatory response. However, the ova recovery rate in NZW rabbits and percentage of fertilized embryos in SC rabbits were significantly affected in the treatment group. The dose of PMSG used might be higher and it would have caused disturbance in terms of recovery as well as the fertilization rate of the embryos. However, no breed influence could be noticed on the above parameters in control rabbits.

Keywords: breed influence, embryo quality, ova recovery, rabbit, superovulation

IPC Code: Int. Cl.⁷ A61K38/24

 

Indian Journal of Biotechnology
Vol. 5, January 2006, pp. 58-61

 

 

 

Phylogentetic analysis of Mycobacterium leprae genome for identification of novel drug targets

Aditya Saxena, Pritish Varadwaj, S Singh, Manoj Jaiswal² , K Misra¹ and T Lahiri^*

The reductive evolution provides a pathogen minimal gene set. Due to loss of genes, for drug target identification, biochemical techniques cannot be used since these pathogens are difficult to culture. Besides classical drug targets i.e. toxins, adhesions, some novel drug targets, using a molecular phylogeny approach, can be identified. Horizontally transferred genes in bacterial pathogen from their eukaryotic host has been classified as ‘Novel drug target’, and a reusable system for identifying horizontally transferred gene has been built using a molecular phylogeny approach. Using this system, two genes have been identified in Mycobacterium leprae, which have also been identified by other methods.

Keywords: horizontal gene transfer, novel drug targets, phylogenetic analysis

IPC Code: Int. Cl.⁷ C12R1:32

 

Indian Journal of Biotechnology
Vol 5, January 2006, pp 62-67

 

 

 

RAPD analysis of genetic variability in Pinus gerardiana Wall. in Kinnaur (Himachal Pradesh)

Anil Kant^*, D Pattanayak¹, S K Chakrabarti¹, Rajan Sharma, Manisha Thakur² and D R Sharma²

RAPD analysis was carried out to determine the genetic diversity of Pinus gerardiana in Himachal Pradesh. Twenty-four genotypes of P. gerardiana growing at different locations in the distribution range of species were sampled randomly for analysis. Thirty random primers generated 413 fragments, of which 390 (94%) were polymorphic in nature. Eleven of the 24 individual genotypes were distinguished by unique bands specific to them. The similarity coefficient values suggested a wide genetic base of genotypes taken in the experiment. An UPGMA dendrogram based on similarity coefficient indices indicated that genotypes did not cluster according to their site of collection. This can be attributed to highly cross-pollinating nature of the species and small distributional range in the area.

Keywords: Chilgoza pine, genetic diversity, Himalayas, Pinus gerardiana, RAPD

IPC Code: Int. Cl.⁷ C12N15/10

Indian Journal of Biotechnology
Vol. 5, January 2006, pp 68-75

 

 

 

Optimization of microbial degradation of an azo dye (methyl red) in fixed film bioreactors

Suresh Kumar, K P Sharma^*, Shweta Sharma¹, Ruby Grover, Pawan Kumar, Pratima Soni and Subhasini Sharma¹

The effects of inorganic (5 ppm PO₄-P and 50 ppm NO₃-N) and organic (30 mL/day milk whey) nutrients were examined on the microbial degradation of 100 ppm methyl red (MR) at one day retention period under aerobic and anaerobic conditions in two types of fixed film bioreactors; one prepared by using only gravel while the other, by using a mixture (3:1) of gravel and coarse river sand. The performance of the bioreactor outflows was judged (with reference to the four types of inflows used) on the basis of their per cent decolourisation, reduction in COD load and toxicity to Lemna, which was found to be more sensitive than Hydrilla. The order of their performance was as follows: MR (100 ppm) +PO₄-P (5 ppm) >/» MR (100 ppm) + PO₄-P+NO₃-N+ Milk whey > MR (100 ppm) + PO₄-P+NO₃-N (50 ppm) >/»MR (100 ppm). In MR +PO₄-P treatment, the outflows from aerated bioreactors were found non-toxic to Lemna and percentage reduction in COD load (63-70%) and decolourisation (90-94%) were also maximum. They, however, were found toxic in other treatments. Among the bioreactors, gravel bed exhibited excellent performance.

Keywords: bioreactor, solid matrix-texture, aerated and non-aerated conditions, inorganic and organic nutrients, methyl red, Bacillus sp., fungi

IPC code:  Int. Cl.⁷ C02F3/34; C02F103:30; C12R1: 645, 1:07, 1:67, 1:68, 1:685, 1:885

 

Indian Journal of Biotechnology

Vol. 5, January 2006, pp. 76-79

 

 

 

Production of PHB (bioplastics) using bio-effluent as substrate by Alcaligens eutrophus

B Senthil Kumar and G Prabakaran¹*

Alcaligenes eutrophus MTCC 1285 produced bioplastics (PHB) in N₂ deficient medium containing carbon (glucose concentration 2%). Using sago effluent, thippi and molasses as substrates and at different pH (6.9-8.0) the production of PHB was recorded; higher production of the biopolymer was obtained at pH 8.0 in the molasses based production media. The amount of PHB produced at pH 6.9 was 0.8, 0.5, 0.4, and 0.73 (g/mL^-1) and 1.1, 0.65, 0.55 and 1.0 (g/mL^-1), respectively in glucose, sago, thippi, molasses substrate based media. The present investigation revealed that bio effluents could be used for the production of PHB so as to decrease the pollution caused by them.

Keywords: PHB, Alcaligens eutrophus, bio-effluent, bioplastics

IPC code: Int. Cl.⁷ A01N63/00; C08G63/00; C12R1:05

 

Indian Journal of Biotechnology
Vol. 5, January 2006, pp 80-83

 

 

 

Optimization of process parameters for alkaline protease production under solid-state fermentation by Thermoactinomyces thalpophilus PEE 14

G Divakar, M Sunitha, P Vasu, P Udaya shanker and P Ellaiah*

Production parameters of extracellular alkaline protease employing our laboratory isolate Thermoactinomyces thalpophilus PEE 14 under solid-state fermentation (SSF) were optimized. Wheat bran the best substrate among the 15 substrates used for optimization, showed the highest activity (1620 PU/g). The physical and chemical parameters were also optimized. The maximum enzyme activity under optimum conditions was obtained with incubation period 72 h, incubation temperature 55ºC, initial pH 10, inoculum level 20%, level of salt solution 2:10 and initial moisture level 80%. Increase of enzyme activity by 60% (2576 PU/g) was observed when compared with the unoptimized conditions.

Keywords: alkaline protease, optimization, SSF, Thermoactinmyces thalpophilus PEE 14, wheat bran

IPC code:   Int. Cl.⁷ C12N9/50; C12R1:01

Indian Journal of Biotechnology

Vol 5, January 2006, pp 84-88

 

 

 

Immobilization of Pleurotus ostreatus 1804 on PUF cubes: Influence of mycelial growth pattern on laccase yield

K Krishna Prasad¹, S Venkata Mohan¹, B R Pati² and P N Sarma^1*

The influence of immobilized Pleurotus ostreatus 1804, on polyurethane foam (PUF) cubes, was investigated on laccase yield as a function of immobilized PUF cubes and their repeated application. Enhanced laccase expression after fermentation was observed with immobilized PUF cubes (3109 U in 168 h) compared to free mycelia (2583 U in 192 h). The expression of laccase during fermentation was monitored by SDS-PAGE electrophoresis, which showed a band with a molecular mass of 63 kDa. Effective laccase yield was also observed with repeated application of immobilized PUF cubes consistently upto 8 batches of fermentation. Scanning electron microscope (SEM) images revealed the pattern of spiraling colonization and aggregation of the P. ostreatus mycelia on PUF cubes. During repeated application, the hyphal growth on the PUF cubes increased consistently, manifested by both the processes of tip extension and branching, permitting the fungal colonization.

Keywords: laccase, immobilization, PUF cubes, mycelial aggregation, SEM imaging, SDS-PAGE electrophoresis

IPC Code: Int. Cl.⁷ A01N63/04; C12N9/00, 11/08; C12R1:645

Indian Journal of Biotechnology
Vol. 5, January 2006, pp 89-93

 

 

 

Characterization of Cucumber mosaic virus infecting Indian long pepper (Piper longum L.) and betel vine (Piper betle L.) in India

P S Hareesh, R Madhubala and A I Bhat*

Natural infection of Cucumber mosaic virus (CMV) in Indian long pepper (Piper longum L.) and betel vine (Piper betle L.) was detected by reverse transcription polymerase chain reaction (RT-PCR). The coat protein gene sequences of CMV infecting these hosts were amplified. The resulting amplicons were cloned and sequenced. In both the cases coat protein gene consisted of 657 nucleotides, potentially encoding for a protein of 218 amino acids. The sequence comparisons revealed 100% identity of these isolates both at amino acid and nucleotide levels, suggesting a common origin. Sequence analyses with various CMV isolates showed that CMV infecting banana and black pepper in India to be more close to isolates from Indian long pepper and betel vine. Based on the coat protein gene sequence identities, it was concluded that CMV infecting both Indian long pepper and betel vine belong to the subgroup I. This is the first report on molecular characterization of CMV infecting Indian long pepper and betel vine.

Keywords: betel vine, coat protein gene, Cucumber mosaic virus, Indian long pepper,

IPC Code: Int. Cl.⁷ C12N7/02, 15/09; C12R1:94

Indian Journal of Biotechnology

Vol 5, January 2006, pp 94-98

 

 

 

Occurrence of Iris mild mosaic potyvirus in cultivated iris in India

Saurabh Kulshrestha, Vipin Hallan, Gaurav Raikhy, Raja Ram, I D Garg¹, Q M R Haq² and A A Zaidi*

Iris (Iris x hollandica Hort. cv. Bluemagic) plants showing mosaic symptoms were tested for the presence of Iris mild mosaic potyvirus (IMMV) by host range, enzyme linked immunosorbent assay (ELISA) using antibodies specific for IMMV), reverse transcription polymerase chain reaction (RT-PCR), immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) using potyvirus group specific primers and antibodies specific to IMMV, immune electron microscopy and cytopathology. Presence of IMMV was detected by ELISA, confirmed by RT-PCR, IC-RT-PCR, IEM, cytopathology and sequencing of the cloned PCR product. From the sequence of the virus, specific primers were designed and specific detection of IMMV from iris was standardized.

Keywords: cytopathology, ELISA, IC-RT-PCR, IEM, Iris mild mosaic potyvirus (IMMV)

IPC Code: Int. Cl.⁷ C07K16/10; C12N15/10; G01N33/53

Indian Journal of Biotechnology
Vol. 5, January 2006, pp. 99-106

 

 

Genotype dependent influence of phytohormone combination and subculturing on micropropagation of sugarcane varieties

Nandita Singh, Anil Kumar and G K Garg*

Eight varieties of sugarcane, viz. Co.89003, Co.91010, Co.96258, Co.97017, Co.P84211, Co.P84212, Co.P90223, and Co.S767, were successfully established and micropropagated using shoots tips as explants. Four combinations of phytohormone, having NAA (1 mg/L) at fixed conc. and two cytokinins, i.e. BAP and Kn, at varying conc., were used; these were designated as A₁ (1 mg/L BAP + 2 mg/L Kn); A₂ (1 mg/L BAP + 3 mg/L Kn); A₃ (3 mg/L BAP + 2 mg/L Kn) and A₄ (6 mg/L BAP and 1 mg/L Kn). All the varieties were successively sub cultured up to 8^th passage. Maximum percentage of explant establishment (80-100%) was observed for all the varieties, except Co.P.84212 and Co.P. 90223 (50-60%). No influence of genotype was found on the days required for side bud induction, average length of shoots from side bud, and number of days for multiple shoots when explants were placed in various phytohormone combinations. However, individual varieties showed preference for higher or lower conc. of cytokinins, i.e. growth of shoots in varieties Co.89003, Co.91010, Co.96258, Co.97017, Co.P84212 and Co.P.90223 was better in A₁ and A₂ hormone profile, while that of Co.91010 and Co.S767 in A₃ and A₄ hormone profile.

Keywords: genotype, micropropagation, phytohormone combinations, Saccharum

IPC Code: Int. Cl.⁷ A01H4/00

 

Indian Journal of Biotechnology
Vol. 5, January 2006, pp 107-111

 

 

 

Callus induction from Ipomoea aquatica Forsk. leaf and its antioxidant activity

K Nagendra Prasad, M Siva Prasad¹, G R Shivamurthy and S M Aradhya^1*

Callus from the leaves of Ipomoea aquatica Forsk. was initiated on Murashige and Skoog’s basal media supplemented with various combinations of auxins 2,4-dichlorophenoxyacetic acid (2,4-D), a-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole butyric acid (IBA) with kinetin/6-benzyl aminopurine (BA). Callus production was observed in all the media but with varied mass. Highest percentage of callus response was obtained in combination of NAA (1.5 mg L^-1) with kinetin (0.5 mg L^-1). The friable callus was white in NAA supplemented and brown in 2,4-D and kinetin supplemented media. Three distinct phases viz., lag, exponential or liner and log phase were observed in the growth of callus. Antioxidant activities were analyzed by DPPH, TBARS and metal chelating method. Hyper antioxidant activity was observed in 1-month-old callus produced by NAA in combination with kinetin. The EC₅₀ value of callus extract was 38 ± 3.05 and 54 ± 3.60 by DPPH and TBARS methods, respectively as against 58 ± 2.6 and 64 ±1.2 in in vivo plant material, however, no metal chelating activity was found in both.

Keywords: antioxidant activity, callus induction, Ipomoea aquatica, leaf explant

IPC Code:  Int. Cl.⁷ A01H4/00; C09K15/00

Indian Journal of Biotechnology
Vol 5, January 2006, pp 112-116

 

 

 

In vitro flowering and rapid propagation of Vitex negundo L.—A medicinal plant

A V Vadawale^1*, D M Barve and ²A M Dave¹

In vitro flowering and an efficient micropropagation protocol was developed for the woody, aromatic and medicinal shrub Vitex negundo (Verbenaceae) by the in vitro culture of nodal segments of mature plant. Murashige and Skoog’s (MS) medium supplemented with 4.4 mM N⁶-benzylaminopurine (BAP) and 0.53 mM α-naphthaleneacetic acid (NAA) were found to develop fully functional flowers. MS medium supplemented with 4.4 mM BAP and 0.53 mM NAA induced an average of five shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. Full strength MS solid medium with 3.69 mM indole-3-butyric acid (IBA) exhibited the best in vitro rooting. Ninety per cent of the rooted shoots survived when transferred to green house and subsequently to the field.

Keywords: inflorescence, medicinal plant, micropropagation, Verbenaceae, Vitex negundo

IPC Code:  Int. Cl.⁷ A01H4/00, 5/02

 

Indian Journal of Biotechnology
Vol. 5, January 2006, pp 117-119

 

 

Short Communications

 

Amplification and cloning of canine parvovirus VP1 gene into mammalian expression vector

P K Gupta, A A Raut and A Rai*

The VP1 gene of canine parvovirus was amplified by polymerase chain reaction using the thermostable polymerase with proof reading activity. Taq DNA polymerase was used to add ‘A’ overhang in the PCR product. This ‘A’ tailed PCR product was cloned in TA cloning vector pTargeT and transformed in Escherichia coli JM109 cells. The recombinant plasmid containing the VP1 gene insert was screened and characterized using restriction enzyme analysis and nested PCR.

Keywords:            Canine parvovirus, capsid protein, VP1 gene, PCR, cloning, plasmid vector

IPC Code:    Int. Cl.⁷ C12N15/09

Indian Journal of Biotechnology

Vol. 5, January 2006, pp. 120-122

 

 

 

Designing specific oligonucleotide primers for metallothionein genes

P J Thomas, T Anand, P Suresh¹, S Janarthanan¹ and S Vincent*

A software program to compute primers for metallothionein (MT) genes has been developed. The program referred to as Gprimer is an algorithm that follows a unique concept of primer selection by identifying the functionally active regions of MT genes. The program has been tested successfully in silico with available MT genes. Gprimer program services can be accessed at the web site: http://www.enviroclub.tk or http://www.freewebs.com/loyolaenviro/gprimer.html.

Keywords: metallothionein (MT) genes, specific primer, algorithm design