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Indian Journal of Biotechnology

 

 

ISSN:0972-5849

 

CODEN:IJBNAR 3(1) 1-144 (2004)

VOLUME 3

NUMBER 1

JANUARY 2004

 

CONTENTS

 

Reviews

 

Topical immunization: Mechanistic insight and novel delivery systems

9

Prem N Gupta, P Singh, V Mishra, Sanyog Jain, P K Dubey and S P Vyas

 

 

 

Gene manipulation in Streptomyces

22

B K Bhattacharyya and S K Sen

 

 

Embryo rescue: A tool to overcome incompatible interspecific hybridization in Gossypium Linn.A review

 

29

S S Mehetre and A R Aher

 

 

 

Papers

 

Detection of virulence in Indian isolates of Salmonella by polymerase chain reaction

37

M K Saxena, V P Singh, B D Lackchura, Anjani Saxena and Bhaskar Sharma

 

 

 

Analysis of genetic relatedness in Gossypium species using RAPD

41

M Gomes, G Kulothungam, S S Mehetre and S Eapen

 

 

 

Screening of mulberry (Morus spp.) for salinity tolerance through in vitro seed germination

 

47

K Vijayan, S P Chakraborti and P D Ghosh

 

 

 

Quantification of urinary oxalate by immobilized oxalate oxidase of forage sorghum leaf

52

Vijay Kalra and C S Pundir

 

 

 

Induction and properties of (13)-b-D-glucanase from Aureobasidium pullulans

58

M S Dake, J P Jadhav and N B Patil

 

 

 

Optimization of process parameters for production of lipase in solid-state fermentation by newly isolated Aspergillus species

 

65

K Adinarayana, K V V S N Bapi Raju, M Iqbal Zargar, R Bhavani Devi, P Jhansi Lakshmi and P Ellaiah

 

 

 

Industrial production of Penicillium corylophilum on rice husk and its application in jute processing

 

70

M K Basak, P K Ganguly, S K Bhaduri and Sutripta Sarkar

 

 

 

Determination of genetic basis for biosurfactant producrtion in distillery and curd whey wastes utilizing Pseudomonas aeruginosa strain BS2

 

74

Kirti Dubey and Asha Juwarkar

 

 

 

Dot-blot assay using reverse-probe genomic hybridization of repetitive DNA for environmental monitoring of fluorescent Pseudomonads

 

82

Reeta Goel, Praveen Kumar, Rashmi Sinha and Sonu Ambwani

 

 

 

Medium optimization for bioleaching of metals from Indian bulk polymetallic concentrate

 

86

D R Tipre, S B Vora and S R Dave

 

 

 

In vitro growth of Tagetes patula L. hairy roots, production of thiophenes and its mosquito larvicidal activity

 

92

T Rajasekaran, G A Ravishankar and B Obul Reddy

 

 

 

Transformation of oilseed mustard Brassica juncea (L.) Czern & Coss cv. Pusajaikisan with snowdrop lectin gene

 

97

Manju Sharma, Rohini Sahani, Rekha Kansal and K R Koundal

 

 

 

High frequency shoot regeneration from Phyllanthus amarus Schum. & Thonn.

103

Kiran S Ghanti, B Govindaraju, R B Venugopal, S Ramgopal Rao, C P Kaviraj, F T Z Jabeen, Arvind Barad and Srinath Rao

 

 

 

Rapid axillary bud proliferation and ex vitro rooting of herbal spice, Mentha piperita L.

108

C Sunandakumari, K P Martin, M Chithra, S Sini and P V Madhusoodanan

 

 

 

Efficient micropropagation of Vanilla planifolia Andr. under influence of thidiazuron, zeatin and coconut milk

 

113

P Giridhar and G A Ravishankar

 

 

 

Factors enhancing somatic embryogenesis and plant regeneration in sugarcane (Saccharum officinarum L.)

 

119

Navdeep K Gill, Raman Gill and S S Gosal

 

 

 

Identification of in vitro responsive immature embryo size for plant regeneration in Sudan grass (Sorghum sudanenses Piper)

 

124

Sanjay Gupta, V K Khanna, Rameshwar Singh and G K Garg

 

 

 

Micropropagation of Eclipta alba Hassk.: An approach to shorten the protocol

128

Archana J Gawde and G T Paratkar

 

 

 

Short Communications

 

Nucleic acid probe based technique for detection of cotton leaf curl virus in India

133

Pradeep Sharma, Narayan Rishi and V G Malathi

 

 

 

Micropropagation of Crataeva magna (Lour.) DC.A medicinal plant

136

A Benniamin, V S Manickam, M Johnson and L H Joseph

 

 

 

Editors Note

139

NISCAIR Policy on Plagiarism

 

 

 

Instructions to Contributors

141

   
Author Index
 

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 9-21

 

Topical immunization: Mechanistic insight
and novel delivery systems

 Prem N Gupta, P Singh, V Mishra, Sanyog Jain, P K Dubey and S P Vyas*

 

Topical application of antigen and adjuvant directly on intact skin, termed as Topical Immunization (TI) or Transcutaneous Immunization (TCI), is a novel and emerging method of vaccine delivery because it is safe and convenient. Moreover, skin is potentially rich site for immunization. Immune response elicited by TI depends upon structure and composition of the skin of target species. TI induces potent, functional immune responses vis--vis offers significant practical advantages for vaccine delivery. Various routes of carrier entry into the skin include the intercellular pathway, the transcorneocyte pathway and the trans-appandageal pathway. Among various approaches for topical immunization, namely physical, chemical and vesicular, latter is gaining wide attention. Vesicular carriers, i.e. liposome, niosome, transfersomes and virosome, elicit immune response by different mechanisms. Some lipids directly lower the skin permeability barrier, which resides primarily in the stratum corneum. Hence, specially designed lipid vesicles, used as topical delivery system, are attracting increasing attention and can be used for TI. The review covers brief immunology of skin and an insight into delivery concepts of topical immunization with emphasis on vesicular systems.

 

Keywords: topical immunization, transcutaneous immunization, novel delivery systems, liposomes, niosomes, transfersomes, ethosomes

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 22-28

 

Gene manipulation in Streptomyces

B K Bhattacharyya1* and S K Sen2

 

The gene cloning and recombination in the genus Streptomyces offers a good possibility to improve secondary metabolite production and creating new antibiotics. This type of genetic manipulation facilitated by the construction of different novel cloning vectors like plasmids, phages and transposable elements and also robust gene cloning methods. This review article describes some of these vectors and regulation of genes in Streptomyces.

 

Keywords: Streptomyces, gene cloning, plasmid, phage, transposon

 

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 29-36

 

Embryo rescue: A tool to overcome incompatible interspecific hybridization in Gossypium Linn.  A review

S S Mehetre* and A R Aher

 

Successful introgression of desirable characters from certain species could not be achieved because of different incompatibility reasons. Such incompatibility can be overcome by ovulo-embryo culture. From the literature reviewed so far, it appears that the technique involving excision of 10 to 15-day-old (after pollination) embryos and their subsequent culture on different combinations of BT and MS media was successful in obtaining difficult interspecific hybrids in Gossypium spp. BT and MS media supplemented with phytohormones, casein hydrolysate and IAA were more effective for culturing interspecific hybrid embryos. Other factors, like age and genotype of embryo; basal media, temperature and photoperiod during culturing; and hardening of plants are also important in determining the success of the interspecific embryo culture in Gossypium spp.

 

Keywords: Gossypium spp., embryo rescue, interspecific

 

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 37-40

 

Detection of virulence in Indian isolates of Salmonella by
polymerase chain reaction

 M K Saxena1*, V P Singh1, B D Lackchura3, Anjani Saxena3 and Bhaskar Sharma2

 

Virulence in Salmonella is reported to be caused by large plasmids of 60-100 kb. In the present study, 24 isolates of four Salmonella serovars (S. Dublin, S. Abortusequi, S. Bareilly and S. Choleraesuis) were studied for their in vivo virulence, plasmid profiling and virulence (Vir) gene detection by PCR. Nineteen isolates were found to be virulent inducing different degrees of mortality in mice. In 5 non-virulent isolates, the large plasmid was absent and there was also no amplification of Vir gene by PCR. It clearly indicates a positive correlation between virulence of the organism and the presence of plasmid and amplification of Vir gene.

 

Keywords: Salmonella, serovars, virulence, plasmid, polymerase chain reaction (PCR), Vir gene

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 41-46

 

Analysis of genetic relatedness in Gossypium
species using RAPD

M Gomes1, G Kulothungan1, S S Mehetre2 and S Eapen1*

 

RAPD markers were employed to assess genetic relatedness in seven Gossypium species, which included G. hirsutum CMS lines and cultivars, G. arboreum GMS lines, cultivars, and wild species, G. raimondii, G. bickii, G. thurberii, G. captis-viridis and G. anomalum, Out of 45 RAPD primers tested, 24 oligonucleotide primers yielded monomorphic amplified products or did not show any amplification product in some of the genotypes. The remaining 21 primers amplified a total of 168 fragments with an average of 9.8 fragments per primer. Out of the 11 genotypes studied, G. arboreum (G 27) produced the maximum number of DNA amplified fragments and G. raimondii produced the lowest number. On the basis of similarity coefficients cluster analysis was performed using UPGMA method. Cluster analysis resulted in 4 main cluster groups. Cluster one contained two subclusters IA and IB. Subcluster IA consisted of G. hirsutum lines, which represent AD genome. Subcluster IB contained G. arboreum genotypes with A genome. Cluster II consisted of G. raimondii, G. thurberii, which belong to the D genome. Cluster III consisted of only G. bickii representing the C genome and cluster 4 consisted of G. anomalum and G. captis-viridis representing the B genome. The clustering pattern obtained using RAPD analysis in the present study is in conformity with available information based on cytogenetic relationship.

 

Keywords : RAPD markers, Gossypium species, genetic diversity

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 47-51

 

Screening of mulberry (Morus spp.) for salinity tolerance through in vitro seed germination

K Vijayan1*, S P Chakraborti and P D Ghosh2

 

Salt tolerant genotypes were identified through screening of the seeds under in vitro conditions. Seeds from 43 promising mulberry genotypes were germinated on MS basal medium in Petri-plates containing 0.0, 0.25, 0.5, 0.75, 1.0, and 1.25% of NaCl (EC 5.3, 9.2, 12.7, 15.4, 20.0 and 25.4 dSm-1 respectively). Seeds from different genotypes showed wide variability to the salinity. Seeds of English black, Rotundiloba, KPG-3, Kolitha-3, Mysore local and Sultanpur showed considerable tolerance to salinity. Wide variability on the behavior of the seeds from genotypes suggests that many genotypes are tolerant to salinity. Thus, these genotypes could be selected for further testing on saline soils under ex vitro conditions. This study offers an added scope of selecting the seedling, which shows tolerance to absolute salinity, and transplanting them through gradual hardening like other tissue cultured plants. Since screening of large number of genotypes under ex vitro conditions entails huge investment and often the interaction of other soil factors make the assessment difficult, screening under in vitro conditions is an attractive alternative, which is more easy, efficient and needs less space and time to screen large number of seeds and genotypes for salinity tolerance in mulberry.

 

Keywords: seed germination, salinity, mulberry, Morus spp

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 52-57

 

Quantification of urinary oxalate by immobilized oxalate oxidase of forage sorghum leaf

Vijay Kalra and C S Pundir*

 

A partially purified oxalate oxidase from leaves of 10-day-old seedlings of forage sorghum was immobilized covalently onto alkylamine glass beads affixed on inner base of a glass beaker. The enzyme retained 5.17% of its initial activity with a conjugation yield of 182 mg/g support. After immobilization, the enzyme showed an increase in its optimum pH and Km for oxalate but slight decrease in incubation temperature and time for maximum activity as compared to free enzyme. The glass beaker bound enzyme was employed for determination of urinary oxalate. The urinary oxalate in apparently healthy persons, as measured by the glass beaker, was found to be in the range of 12.5 to 29.7 mg/l (mean 20.9 mg/l) for females and 25.7 to 45.4 mg/l urine (mean 37.2 mg/l) for males. The glass beaker provided 70 reuses of immobilized enzyme with ease in handling.

 

Keywords: oxalate, oxalate oxidase, immobilization, forage sorghum, urine, alkylamine glass

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 58-64

 

Induction and properties of (13)-b-D-glucanase from Aureobasidium pullulans

M S Dake*, J P Jadhav and N B Patil

 

The structure determination of polysaccharides using specific enzyme(s) as a tool has been of great importance. Such an enzyme, (13)-b-D-glucanase was induced in Aureobasidium pullulans by yeast b-glucan as a potent inducer. The formation of enzyme was, however, repressed by glucose. The purified enzyme has molecular weight 230 kDa, optimum pH 5.5 and optimum temperature 60C. The enzyme hydrolyzes laminarin and yeast b-glucan. The km value obtained for laminarin was 0.17%. The analysis of the hydrolyzed product demonstrates that the enzyme acts on b (1,3) linkages from the non-reducing end(s).

 

Keywords: Aureobasidium pullulans, induction, glucanase, purification, extracellular enzymes, b-glucan

  

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp 65-69

 

Optimization of process parameters for production of lipase in solid-state fermentation by newly isolated Aspergillus species

K Adinarayana, K V V S N Bapi Raju, M Iqbal Zargar,

R Bhavani Devi, P Jhansi Lakshmi and P Ellaiah*

 

Of the 34 fungal species, isolated from a number of oily substrates, 9 exhibited lipase activity. AU 15, identified as Aspergillus sp., was found to be excellent lipase producer in submerged fermentation and was selected for solid-state fermentation (SSF). Among substrates like oil cakes of coconut, groundnut and sesame, wheat rawa, bombay rawa and soya beans (crushed), wheat rawa showed the highest lipase activity. The maximum enzyme yield (1934 U/g) was obtained with basal medium containing wheat rawa, olive oil and corn steep liquor, at 80% moisture content, pH 7.0 and 96 hrs incubation.

 

Keywords: lipase; Aspergillus species; solid state fermentation (SSF); submerged fermentation (SmF)

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 70-73

 

Industrial production of Penicillium corylophilum on rice husk and its application in jute processing

M K Basak, P K Ganguly, S K Bhaduri* and Sutripta Sarkar

 

Low quality jute bark, after treatment with a fungal culture of Penicillium corylophilum, showed improvement in fibre quality viz. strength and fineness and enhanced processability of the yarn at mill level. Conventional potato dextrose broth (PDB) was used for large-scale production of the fungus for application on barky jute at the jute mill. But, the process has several limitations like non-uniform growth of fungus and high cost for labour and energy inputs as well as for substrate. To overcome the limitations, the fungus was produced using rice husk, a cheap agro-residue, as substrate. Rice husk as substrate provided good and uniform growth of fungus, which sustained for at least 90 days. The fungus produced on rice husk has been used successfully for processing of low quality jute fibre in jute mills. There was overall improvement in spinning parameter due to application of the culture.

 

Keywords: Penicillium corylophilum, rice husk, solid state fermentation, jute processing, mill trial

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 74-81

 

Determination of genetic basis for biosurfactant production in distillery and curd whey wastes utilizing Pseudomonas aeruginosa strain BS2

Kirti Dubey and Asha Juwarkar*

 

Pseudomonas aeruginosa strain BS2 has been demonstrated to have an ability to produce potent biosurfactant, an eco-friendly substitute to synthetic surfactants from distillery and whey wastes and capable of reducing the pollution load of these wastes in the range of 85-90%. To determine the basis for future identification of the genes responsible for biosurfactant production from wastes, studies on the presence of plasmid if any, its profile and role in biosurfactant production were performed. Suitable plasmid screening technique was selected because strain BS2 produced excessive slime in Luria Burnetti broth, which interfered with the migration and detection of plasmid. Among the several methods, alkaline lysis method was the most suitable which aided in recovery of slime-free cell lysate and resulted in the formation of a discrete band of plasmid in agarose gel. Plasmid profile study demonstrated that plasmid had high molecular weight of 32.08106 Da and possessed the genetic determinants for antibiotics (chloramphenicol, tetracycline and sulphonamide) and heavy metal salt (mercuric chloride) resistance and were used as markers in curing experiment. To determine the role of megaplasmid in biosurfactant production, curing of megaplasmid was performed at highest sublethal doses of acridine orange (100 g/ml) and mitomycin-C (15 g/ml). Results indicated that only mitomycin-C treatment resulted in 28% of cell population which turned sensitive towards marker antibiotics and heavy metal salt due to loss of megaplasmid, which was further confirmed by agarose gel electrophoresis. Comparative analysis of biosurfactant production potential of cured cells with that of wild cells in both the wastes showed that the cured cells had similar potential capability of biosurfactant production as of wild strain which illustrates that genes responsible for biosurfactant production in distillery and whey wastes utilizing strain BS2 were not plasmid borne but resided on the chromosome where they are more stable.

 

Keywords: biosurfactant, curd whey, curing, distillery waste, plasmid, Pseudomonas aeruginosa strain BS2

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 82-85

 

Dot-blot assay using reverse-probe genomic hybridization
of repetitive DNA for environmental monitoring of
fluorescent Pseudomonads

Reeta Goel*, Praveen Kumar, Rashmi Sinha and Sonu Ambwani

 

The ability of fluorescent pseudomonads to produce antimicrobial metabolites, siderophores and plant hormones makes them an important biocontrol agent as well as plant growth promotary rhizobacteria. For an efficient and potential use as bioinoculant, their quick, specific and sensitive monitoring in environmental sample is essential. A quick detection protocol has been proposed for Pseudomonas fluorescens in soil samples. The approach involved shotgun cloning of restricted genomic DNA with subsequent selection of cloned repetitive DNA by reverse-probe genomic hybridization. In which the plasmid DNA clones were probed with labelled genomic DNA. Further, plasmid DNA with repetitive sequences was used as probe for highly sensitive and specific detection of the soil rhizobacteria.

 

Keywords: Pseudomonas fluorescens, reverse-probe genomic hybridization, dot-blot assay, soil DNA, repetitive sequences

 

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 86-91

 

Medium optimization for bioleaching of metals from Indian bulk polymetallic concentrate

D R Tipre1, S B Vora2 and S R Dave1*

 

Extensive bioleaching work is mainly done only for the Cu and gold extraction from concentrates.  Bioleaching of bulk concentrate is at the developing stage. The present work deals with the formulation of an economic medium for the bioextraction of metals from GMDC polymetallic bulk concentrate. For the medium formulation, a 24 factorial statistically designed experiment was adapted. Effect of concentration at two levels each of K2HPO4, (NH4)2SO4, KCl and inoculum was studied. For Cu and Zn extraction, inoculum was the most significant factor with the nutrients checked. KCl and its interaction were found negatively significant. The formulated medium was compared with the 9K- and Jordan's medium. The highest Cu (77.8%) and Zn (84%) extraction achieved with the formulated medium was 1.25 and 1.32 folds higher than the universally used 9K- medium. The final metal extraction rates obtained were 0.65 and 4.2 g/l/d for Cu and Zn respectively with the designed medium. The composed medium was at least 4 times cheaper, less acid consuming and more suitable for the metal extraction from GMDC polymetallic concentrate compared to 9K- medium.

 

Keywords: bioleaching, polymetallic concentrate, Cu, Zn, Thiobacillus ferrooxidans, factorial

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 92-96

 

In vitro growth of Tagetes patula L. hairy roots, production of thiophenes and its mosquito larvicidal activity

T Rajasekaran, G A Ravishankar* and B Obul Reddy

 

Hairy root culture of Tagetes patula was studied for thiophene production. Growth parameters (on fresh and dry wt basis) were measured for biomass production. The analysis of thiophene was carried out by the methods of Flame Ionization Detection (FID) and mass spectral analysis (GC-MS). The separation profile of the thiophenes indicated the presence of several structurally different thiophenes, predominantly α-terthienyl, which was confirmed by FID and GC-MS analysis. The maximum accumulation of biomass (0.27g.  dry wt/ culture) was recorded on 12th day and thiophene content (0.064%) was recorded as maximum on 9th day. The thiophene produced in hairy roots of T. patula showed larvicidal effect against mosquito larvae.

 

Key words: Tagetes patula, root culture, thiophene, larvicidal activity

  

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 97-102

 

Transformation of oilseed mustard Brassica juncea (L.) Czern & Coss cv. Pusajaikisan with snowdrop lectin gene

Manju Sharma, Rohini Sahni, Rekha Kansal and K R Koundal*

 

An efficient protocol has been developed to transfer snowdrop lectin gene (gna) to Brassica juncea (L.) Czern & Coss cv. Pusajaikisan through Agrobacterium tumefaciens GV 2260 mediated transformation. High frequency regeneration of transformed plantlets has been achieved by using stem segments as explants. Analysis of the putative transformants by PCR amplification and Southern hybridization of genomic DNA showed the successful integration of the transgene in the nuclear genome. The transgenic plants will be further tested for bioassay in future.

 

Keywords: Brassica juncea, Agrobacterium tumefaciens, snowdrop lectin gene, stem segments, transformation

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp 103-107

 

High frequency shoot regeneration from
Phyllanthus amarus Schum. & Thonn.

Kiran S Ghanti, B Govindaraju, R B Venugopal, S Ramgopal Rao, C P Kaviraj,

F T Z Jabeen, Arvind Barad and Srinath Rao*

 

Shoot tip, nodal and internodal segments of Phyllanthus amarus Schum. & Thonn. were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyl amino purine (BAP) and kinetin (Kn) with 15% coconut milk. All the explants responded for regeneration. High frequency (100%) and maximum number of shoots (18.30 0.47) were recorded on medium containing BAP (0.5 mg/l) in shoot tip culture. In vitro shoots were excised from shoot clumps and transferred to rooting medium containing different concentrations of indole butyric acid (IBA) or naphthalene acetic acid (NAA). Maximum number of roots was induced in medium containing IBA (0.5 mg/l). Rooted plantlets were hardened on MS basal liquid medium and subsequently in sterile soil + vermiculite (1:1). Plantlets thus developed were successfully established and finally transferred to greenhouse. The survival rate of the plantlets in the field was found to be very high (85%).

 

Keywords: Phyllanthus amarus, micropropagation, multiple shoots, rhizogenesis

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 108-112

 

Rapid axillary bud proliferation and ex vitro rooting of
herbal spice, Mentha piperita L.

C Sunandakumari1, K P Martin2, M Chithra1, S Sini1 and P V Madhusoodanan1*

 

Efficient protocol for rapid multiplication of the herbal spice Mentha piperita L. through axillary bud multiplication and ex vitro rooting was established using Murashige and Skoog (MS) medium.  Media prepared with tap water and commercial sugar, and those prepared with double distilled water and tissue culture grade sucrose did not show significant difference in the in vitro induction of shoots/node, and roots/shoot. MS medium fortified with 4.44 mM N6-benzyladenine (BA) and 2.32 mM kinetin (Kn) was the best for proliferation of shoots; induced a mean of 4.1 shoots/node explant. The shoots attained a height of more than 4.5 cm bearing more than 5 nodes within 40 days. Excision and culture of in vitro derived node segments on medium with 3.33 mM BA and 2.32 mM Kn facilitated enhanced axillary bud proliferation.  Shoots developed were rooted best on half-strength MS medium with 0.49 mM indole-3-butyric acid (IBA); induced a mean of 10.3 roots. In vitro rooted shoots exhibited 100% survival in field conditions. Dipping of the basal end of shoots harvested from multiplication medium in 0.49 mM IBA solution for 10 days induced a mean of 8 roots and its transfer to small pots facilitated survival of 95% rooted shoots. Ex vitro rooting by direct transfer of the shoots from multiplication medium to small pots showed 72% survival. Commercial sugar and tap water and ex vitro rooting make the protocol economic.

 

Keywords: commercial grade sugar, ex vitro rooting, medicinal plant, tap water; Mentha piperita

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 113-118

 

Efficient micropropagation of Vanilla planifolia Andr. under influence of thidiazuron, zeatin and coconut milk

P Giridhar and G A Ravishankar*

 

Vanilla planifolia multiplication through tissue culture was worked out with new leads in elucidation of synergistic activity of zeatin, synthetic cytokinin thidiazuron (TDZ) and coconut milk (CM) as well as  N6-benzyladenine(BA) . Multiple shoots were developed from axillary bud explants using Murashige and Skoog (MS) medium supplemented with zeatin, BA+zeatin , TDZ and TDZ+ coconut milk (CM). The nature of explant and the method of explant inoculation onto the medium influence not only multiple shoot production, but also bulbous shoot buds (BS) formation. Zeatin  supported the growth of mostly single shoots, whereas formation of BS was induced in zeatin and BA combination and also in media supplemented with TDZ+10% CM. Subsequent transfer of these BS onto shoot proliferation medium supplemented with BA (8.87 mM) or α-naphthalene acetic acid (NAA) (2.69 mM) resulted in multiple shoot proliferation 17 2.5 shoots and 30 2.1 shoots respectively per explant. The multiple shoots so obtained were transferred to Nitsch medium (N69) containing BA (2.22 mM) and gibberellic acid (GA3 ) (0.029 mM) and also onto simultaneous shoot multiplication and root forming medium for further growth. For the first time the influence of zeatin, TDZ  and coconut milk on shoot multiplication was studied. This protocol is effective in producing micropropagated vanilla plants with successful hardening and field transfer.

 

Keywords: axillary bud, bulbous shoot buds, explant, gibberilic acid, plantlets, rooting

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 119-123

 

Factors enhancing somatic embryogenesis and plant regeneration in sugarcane (Saccharum officinarum L.)

 Navdeep K Gill, Raman Gill and S S Gosal*

 

Factors affecting somatic embryogenesis and subsequent plant regeneration in vitro in sugarcane cultures were highly genotype specific. They were affected by medium constitution, auxins, sugars, amino acids, growth retardants, and desiccation, etc. Supplementation of the control medium independently with maltose (30g/l), proline (560mg/l), abscissic acid [ABA] (2mg/l) and reduced auxin ie 2,4-Dichlorophenoxyacetic acid [2,4-D] (3mg/l) enhanced somatic embryogenesis considerably. Similarly, higher shoot regeneration was achieved on a medium containing only cytokinin ie 6-Benzylaminopurine [BAP] (0.5mg/l). Addition of ABA (2mg/l), proline (560mg/l) and dessicating conditions, created by increasing agar concentration, also increased shoot regeneration. Rooting was induced completely in the rooting medium containing only α-naphthalene acetic acid [NAA] (5mg/l) rather than a combination of indole butyric acid [IBA] (2mg/l) and NAA (3mg/l) both accompanied by high sugar ie sucrose (7%).

 

Keywords: somatic embryogenesis, plant regeneration, maltose, proline, dessication, auxin, sugarcane

 

 

 

Indian Journal of Biotechnology

Vol. 3, January 2004, pp. 124-127

 

Identification of in vitro responsive immature embryo size for plant regeneration in Sudan grass (Sorghum sudanenses Piper)

Sanjay Gupta1*, V K Khanna2, Rameshwar Singh2 and G K Garg3

 

Immature embryos of various sizes from six genotypes of sudan grass (Sorghum sudanenses Piper) were cultured to identify the best in vitro responsive embryo size. The immature embryos size influenced callus formation (days to callus initiation, callus induction frequency, callus growth) and plant regeneration (shoot induction frequency, shoots per callus) parameters. Immature embryos (0.7-1.5 mm) were quicker to initiate callus, induced fastly growing callus in higher frequency and regenerated shoots more frequently and intensely than immature embryos (1.6-2.5 mm) because of the initiation of callus from rapidly dividing scuttelar cells. Frequent germination and initiation of non-embryogenic callus from plumule or radicle were the main reasons for poor callus induction and plant regeneration responses from 1.6-2.5 mm size immature embryos. Thus immature embryos of 0.7-1.5 mm size may be used for embryogenic culture initiation and plant regeneration.

 

Keywords: sorghum, Sorghum sudanenses, immature embryos, immature embryo size, callus induction, plant regeneration

 

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp.128-132

 

Micropropagation of Eclipta alba Hassk.: An approach to shorten the protocol

Archana J Gawde and G T Paratkar*

 

A successful attempt was made to shorten the protocol for micropropagation of the elite variety of Eclipta alba. Multiple shoots were obtained from nodal explants on MS medium supplemented with different concentrations (0.44-22.2 M) of 6 benzyladenine (BA); the best response was obtained with 4.44 M. Further, the shoot multiplication and simultaneous rooting was obtained with lowered BA concentration (0.44 M). The in vitro plantlets were successfully acclimatized and transferred to the field.

 

Keywords: Eclipta alba, micropropagation, Knops hydroponic solution, vermicompost

 

 

 

Indian Journal of Biotechnology

Vol 3, January 2004, pp. 133-135

 

Nucleic acid probe based technique for detection of
cotton leaf curl virus in India

 Pradeep Sharma1*, Narayan Rishi1 and V G Malathi2

 

Cotton leaf curl disease, caused by a geminivirus, has been detected using viral nucleic acid based hybridization tests in infected hosts and weeds. Cotton leaf curl was transmitted by whitefly, Bemisia tabaci to Gossypium hirsutum varieties, which showed typical symptoms of the disease. Hybridization with [ά-32P] dCTP radiolabelled CLCuV-DNA-A probe, detection of viral nucleic acids in different cotton cultivars viz., HS-6, H-1098, F-846, H-777, H-182, LH-1556 and RST-9 grown under glasshouse conditions, samples collected from Hisar, Sirsa and Dabwali and six weed hosts was carried out. Nucleic acid based tests could therefore be useful in screening of cotton germplasm in cotton breeding programme to detect minor infections.

 

Keywords: diagnostics, geminivirus, cotton, plant virus disease

 

 

 

Indian Journal of Biotechnology

 Vol.3, January 2004, pp. 136-138

 

Micropropagation of Crataeva magna (Lour.)
DC.A medicinal plant

A Benniamin, V S Manickam*, M Johnson and L H Joseph

 

Rapid multiplication of C. magna, a medicinal plant, was achieved by culturing nodal segments on Murashige and Skoogs (MS) medium supplemented with sucrose (3%) and different concentrations of benzyl amino purine (BAP). Nodal segments cultured on MS medium supplemented with 8.8 M of BAP produced multiple shoots (4.4+0.09) with maximum length (63.2+0.92 mm). Rooting of the excised shoots cultured on 9.84 M of IBA in combination with 0.54 M of NAA in half strength MS medium was found suitable. Rooted plants established with 68% success rate in pots after hardening.

 

KeywordsCrataeva magna, in vitro multiplication, nodal segments

 

 

AUTHOR INDEX

 

 

Adinarayana K

65

Goel R

82

Paratkar G T

128

Aher A R

29

Gomes M

41

Patil N B

58

Ambwani S

82

Govindaraju B

103

Pundir C S

52

 

 

Gosal S S

119

Rajasekaran T

92

Bapi Raju K V V S N

65

Gupta P N

9

Rao S

103

Barad A

103

Gupta S

124

Rao S R

103

Basak M K

70

 

 

Ravishankar G A

92, 113

Benniamin A

136

Jabeen F T Z

103

Reddy B Obul

92

Bhaduri S K

70

Jadhav J P

58

Rishi N

133

Bhattacharyya B K

22

Jain S

9

 

 

Bhavani Devi R

65

Johnson M

136

Sahani R

97

 

 

Joseph L H

136

Sarkar S

70

Chakraborti S P

47

Juwarkar A

74

Saxena A

37

Chithra M

108

 

 

Saxena M K

37

 

 

Kalra V

52

Sen S K

22

Dake M S

58

Kansal R

97

Sharma B

37

Dave S R

86

Kaviraj C P

103

Sharma M

97

Dubey K

74

Khanna V K

124

Sharma P

133

Dubey P K

9

Koundal K R

97

Singh P

9

 

 

Kulothungam G

41

Singh R

124

Eapen S

41

Kumar P

82

Singh V P

37

Ellaiah P

65

 

 

Sinha R

82

 

 

Lackchura B D

37

Sini S

108

Ganguly P K

70

Lakshmi P J

65

Sunandakumari C

108

Garg G K

124

 

 

 

 

Gawde A J

128

Madhusoodanan P V

108

Tipre D R

86

Ghanti K S

103

Malathi V G

133

 

 

Ghosh P D

47

Manickam V S

136

Venugopal R B

103

Gill N K

119

Martin K P

108

Vijayan K

47

Gill R

119

Mehetre S S

29, 41

Vora S B

86

Giridhar P

113

Mishra V

9

Vyas S P

9

 

 

 

 

 

 

 

 

 

 

Zargar M I

65