Indian Journal of Biotechnology

 

VOLUME 1

NUMBER 3

JULY 2002

 

CONTENTS

Reviews

 

Abiotic stress related genes and their role in conferring resistance in plants

225

        M Z Abdin, R U Rehman, M Israr, P S Srivastava & K C Bansal

 

 

Microarrays—‘Chipping’ in genomics!

245

        Biju Joseph, Anupama Shrinivasan & Govindasamy Kumaramanickavel

 

 

Papers

 

Cultivation of Spirulina in gas induced photobioreactor and isolation of phycobiliproteins

255

        Harshal H Kshirsagar, Madhavi S Revankar, Madhusudan Y Kamat & Smita S Lele

 

 

Steady state performance and sensitivity of a continuous recombinant fermentation with plasmid copy number multiplicity

263

        Pratap R Patnaik

 

Enhancement in inulinase production by mutagenesis in Penicillium purpurogenum

270

        Arun Dev Sharma, Jagpreet Singh Nanda, Prabhjot Kaur Gill,

        Sukdev Singh Bhullar, Prabhjeet Singh & Dhiraj Vyas

 

 

LacZ tagging of phosphate solubilizing Pseudomonas striata for rhizosphere colonization

275

        Shalini Mourya & K S Jauhri

 

 

Optimal exploitation of native arbuscular and vesicular-arbuscular mycorrhizae for improving the yield of banana through IPNM

280

        N V Phirke, S B Chincholkar & R M Kothari

 

 

Micropropagation of elite cultivars of rose-scented geranium (Pelargonium graveolens L’ Herit.) for industrial production of propagules

286

        Ritika Gupta, Shiv Kumar Gupta, S Banerjee, G R Mallavarapu & Sushil Kumar

 

 

Comparison of ELISA and GC methods to detect DDT residues in water samples

292

        B E Amitarani, Akmal Pasha, Putte Gowda, T R Nagendraprasad & N G K Karanth

 

Short Communications

 

Agrobacterium-mediated transformation of Lucerne (Medicago sativa Linn.): Optimizing biological and physical parameters

298

        Suresh Kumar, Vishnu Bhat, B V Bhat & M G Gupta

 

 

In vitro regeneration and hydroxycitric acid production in tissue cultures of
Garcinia indica Chois.

 

301

        Manik D Kulkarni & Manjushri A Deodhar

 

 

Production of plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) in callus and cell suspension cultures of Plumbago indica Linn.

305

        K Satheeshkumar & S Seeni

 

 

News Scan

 

Nanotechnology in biomedical applications

309

        Onkar J Chakre

 

 

Announcements

 

312

Instructions to Contributors

313

 

 

 

Abiotic Stress Related Genes and their Role in Conferring Resistance in Plants

M Z Abdin, R U Rehman, M Israr, P S Srivastava and K C Bansal

 

Abiotic environmental stresses, which limit the plant distribution and productivity, include low and high temperature, salinity and water deficit. Over the last century human activities have increased the level of environmental stress in the form of pollutants such as ozone and heavy metals, levels of UV light reaching the biosphere and salinity in irrigated areas. Plants are sessile and have, therefore, developed mechanisms to survive under extreme environments sometimes in vegetative stages of their life cycle. The recent surge of information on regulation of gene expression under stress conditions as well as the biochemical function of individual proteins in conferring tolerance to stress will help in isolating the genes of interest to produce desired transgenics. The understanding of molecular basis of these survival mechanisms discussed in this review may ultimately help enhance plant productivity in current marginal areas. The review deals with effect of drought and salt stress on plants and the regulatory mechanisms.

Keywords: Arabidopsis, abiotic stress, stress inducible genes, stress resistance, transgenics

 

 

 

Microarrays — ‘Chipping’ in Genomics!

 

Biju Joseph, Anupama Shrinivasan and Govindasamy Kumaramanickavel

 

DNA microarrays or gene chips are an assemblage of assorted short sequences of DNA or polypeptides embedded onto a solid medium such as glass or plastic slides/silicon wafers or nylon membranes. A state of the art technology in the elucidation of molecular basis of gene expressions, this versatile tool finds many applications ranging from assessing the burgeoning sequential information generated by the human genome project, to the grading of tumours and evaluating clinical prognosis. The present review elaborates the historical background of DNA microarrays, their construction as well as discusses with relevant examples, their immense potential in diagnostic and therapeutic research and the drawbacks encountered in their application.

Keywords:cancer biology, DNA microarrays, DNA chips

 

 

Cultivation of Spirulina in Gas Induced Photobioreactor and Isolation of Phycobiliproteins

Harshal H Kshirsagar, Madhavi S Revankar, Madhusudan Y Kamat and Smita S Lele

 

A novel design of Gas Induced Mechanically Agitated Photobioreactor (GIMAP) using the principle of hollow shaft impeller to sparge air is tested with Spirulina as model system. Gas induced impeller is a lesser-explored type of mechanically agitated contactor. It possesses unique characteristics like no separate power requirement for the sparging of the gas, moderate values of mass transfer coefficient and relatively low values of shear stress. These characteristics make it suitable as a photobioreactor although it may not be the best choice for the classical fermentations. GIMAP exhibited good performance giving biomass concentration and high specific growth rate of 4.44 g/l and 1.33 d-1 , respectively. Hydrodynamic stress developed in GIMAP was found to have favourable effect that resulted in production of biomass with more permeable cell walls. As a result it was possible to extract phycobiliproteins without cell rupture by simply changing the pH of the buffer. Further it was possible to recover 23% of phycobiliproteins (92% yield) from the GIMAP culture along with a by-product namely exhaust cell mass with 25% proteins. Simpler downstream processing, additional byproduct coupled and higher growth rates of Spirulina than the conventional cultivation methods indicate the commercial potential of the proposed GIMAP design as photobioreactor.

Keywords: Spirulina, phycobiliproteins, microalgae, photobioreactor, gas-inducing impellers

 

 

Steady State Performance and Sensitivity of a Continuous Recombinant Fermentation with Plasmid Copy Number Multiplicity**

Pratap R Patnaik

 

Fermentations utilizing genetically modified cells usually contain two or more fractions of plasmid-bearing cells differing in the plasmid copy number. Competition among these cells and those without the plasmid may lead to a different performance than in the more 'ideal' situation where all recombinant cells have the same number of plasmids. A recent study considered the steady state sensitivities to the feed concentration of substrate and the dilution rate for a continuous fermentation with Saccharomyces cerevisiae XK1-C2 harbouring the plasmid pSXR125, which synthesizes b-galactosidase, with a uniform copy number. A similar analysis has been applied here to the same fermentation with a binomial distribution in the copy number. Although the steady states and their sensitivities are different from those for a uniform distribution, the dilution rate is still the favoured manipulated variable for control. While a selective medium generates a superior performance, its higher sensitivities require rapid on-line monitoring and anticipatory control.

Keywords: continuous fermentation, plasmid copy number, steady state, sensitivity

 

Enhancement in Inulinase Production by Mutagenesis in Penicillium purpurogenum

Arun Dev Sharma, Jagpreet Singh Nanda, Prabhjot Kaur Gill, Sukdev Singh Bhullar, Prabhjeet Singh and Dhiraj Vyas

Extracellular inulinase producing strain of Penicillium purpurogenum was isolated from rhizosphere soil of chicory. Conidia of this selected strain were subjected to mutagenesis with UV and NTG (3-nitro, 5-methyl guanidine). After mutagenesis, of various colonies screened, few were selected to carry out the inulinase study. The study revealed 2.5-fold higher inulinase activity with 8 to 21-fold I/S ratio in all the selected colonies, indicating the enhancement in inulinase production after both mutagenic treatments in all the selected mutants.

Keywords: Penicillium purpurogenum, I/S ratio, inulinase, invertase, mutants

 

LacZ Tagging of Phosphate Solubilizing Pseudomonas striata for Rhizosphere Colonization

Shalini Mourya and K S Jauhri

 

The Tn5-lacZ marker was used for detection of phosphobacteria, Pseudomonas striata (P-27) in the soybean rhizosphere. The chromogenic marker lacZ (structural gene for b-galactosidase) was introduced into P. striata (P-27) by transposition of Tn5-lacZ from Escherichia coli (S17-1). The mutants, which expressed b-galactosidase activity on selective media seeded with X-gal and IPTG, were screened for P-solubilization and IAA production. Mutant strains (lacZ marked) designated as superior, inferior and isogenic to wild type (P-27) with respect to P-solubilization (T-80, T-125, T-128) and IAA production (T-49, T-57, T-87) were inoculated to soybean in a pot experiment.  The inoculated strains were recovered from the rhizosphere at different periods of plant growth by plating on selective medium using lac+ phenotype. A comparison between various mutants in terms of their abilities to colonize the soybean rhizosphere revealed that lacZ insertion or mutational over-expression of plant growth promoting traits did not affect the establishment, population dynamics and ecological fitness of phosphobacteria. The technique of monitoring the tagged strains by direct plating on selective medium was found to be superior compared to conventional techniques.

Keywords: phosphobacteria, Tn5-lacZ marker, ecological monitoring

 

Optimal Exploitation of Native Arbuscular and Vesicular-Arbuscular Mycorrhizae for Improving the Yield of Banana through IPNM

N V Phirke, S B Chincholkar and R M Kothari

 

The R & D banana orchard of the North Maharashtra University harboured diversified species of vesicular-arbuscular mycorrhizal (VAM) fungi belonging to the genera Glomus, Gigaspora, Sclerocystis and Scutellospora. These fungi infected the roots severely (78.3%) and showed elevated (68.7 g-1 soil) spore density in the mycorrhizosphere of plants for integrated plant nutrition management (IPNM) system as compared to chemical fertilizers alone (traditional farming; 49.2% & 39.3 g-1 soil). This resulted in retardation of the establishment of nematodes in root zone of IPNM treatment (2.1 g-1 soil) as compared to chemical treatment (5.5 g-1 soil). VAM fungi enabled proper nourishment and protection of crop in IPNM treatment as compared to traditional way, which was evident from (a) reduced plant mortality (12%) of planted sword suckers, (b) enhanced greenery (foliar chlorophyll 778 mg g-1), (c) improved nutrient uptake on dry weight basis (3.62% N; 0.31% P; & 6.45% K), (d) elevated finger number (137.6 per bunch) size (length 21.6 cm & girth 12.4 cm) and weight (130.8 g), and (e) overall vigour and robustness. Hence, exploitation of VAM through adoption of IPNM system revealed the (i) relatively increased banana productivity (76.5%, 72 tonnes ha-1) and recyclable biowaste (36.6 kg), (ii) saving of 50% chemical fertilizers, and (iii) permitted control over soil fertility in farmer’s favour over traditional cultivation practices. These findings related to native VAM fungi in IPNM, traditional farming and typical soil systems for cultivation of bananas are discussed in detail.

Keywords: improved banana productivity, VAM, IPNM, traditional farming, yield improvement, relative banana productivity

 

Micropropagation of Elite Cultivars of Rose-scented Geranium (Pelargonium graveolens L’ Herit.) for Industrial Production of Propagules

Ritika Gupta, Shiv Kumar Gupta, S Banerjee, G R Mallavarapu and Sushil Kumar

 

Experiments were carried out to develop an efficient micropropagation procedure to produce rooted plantlets from stem and leaf explants of the elite cultivars of rose-scented geranium, Pelargonium graveolens. A set of 8 Murashige and Skoog (MS) salts based media were evaluated for eliciting adventitious shoot regeneration response from stem and leaf explants of three cultivars namely ‘Hemanti’, ‘Bipuli’ and ‘Kunti’. Stem and leaf explants without callus intervention produced 20 or more shoots in about 4 weeks. The directly regenerated shoot mass upon separation underwent good level of rooting on half strength MS medium. Plantlets thus obtained were planted in the field along with their vegetatively propagated control plants. In vitro raised plants were morphologically identical to control. Genetic fidelity of regenerants was further confirmed by gas chromatography of essential oil from aerial parts. Thus, an efficient procedure has become available for the multiplication of elite plant material of P. graveolens required for the intensive cropping in semi-temperate to tropical agro-climates of Indian hills and plains.

Keywords: adventitious shoot proliferation, plantlet multiplication, geranium essential oil, in vitro propagation, geranium planting material

 

Comparison of ELISA and GC Methods to Detect DDT Residues in Water Samples

B E Amitarani, Akmal Pasha, Putte Gowda, T R Nagendraprasad and N G K Karanth

 

ELISA and GC methods were used to analyse DDT residues in about 30 water samples collected from different talukas of Mandya District of Karnataka. Polyclonal antibody based immunoassay developed at CFTRI, Mysore, performed well to detect the DDT residues. The minimum detectable level of DDT by ELISA was one part per billion (ppb) in the water samples tested. The insecticide residue ranged from 1 to 20 ppb. Experiments also revealed no matrix effect and hence did not require any prior clean-up. The pH of the water did not interfere in the assay.  The ELISA method validated in the present work is specific to DDT.   The results of ELISA with respect to DDT residues were found to be comparable to values obtained from the GC analysis of the water samples. The water samples could be directly used for ELISA test, thereby making   the analysis quick, simple and cost effective.

Keywords: DDT residues, ELISA, GC, water samples

 

 

Agrobacterium-mediated Transformation of Lucerne (Medicago sativa Linn.): Optimizing Biological and Physical Parameters

 

Suresh Kumar, Vishnu Bhat, B V Bhat and M G Gupta

 

An optimized protocol for the transformation of Indian cultivars of lucerne (Medicago sativa Linn.) using Agrobacterium tumefaciens has been developed. It involved a selectable marker gene encoding hygromycin phosphotransferase (hpt II) which confers resistance to the antibiotic hygromycin, together with a reporter gene (uid A) encoding b-D glucuronidase (GUS); cotyledonary and hypocotyl explants from in vitro grown 5 days -old seedlings raised from mature seeds of genotype L-2 and IL-75 as target tissues, and co-cultivation of explants with A. tumefaciens strain LBA-4404 harbouring plasmid pCAMBIA-1301. Cotyledonary explant was found to be more responsive for transformation. Bacterial density of 5´109 cells/ml and co-cultivation for three days were found to be most effective for stable GUS expression in 91% of the hygromycin-resistant calli.

Keywords: lucerne, Agrobacterium, genetic transformation, forage quality improvement

 

In Vitro Regeneration and Hydroxycitric Acid Production in Tissue Cultures of Garcinia indica Chois.

Manik D Kulkarni and Manjushri A Deodhar

Garcinia indica is a rich source of hydroxycitric acid (HCA), an important biological active plant metabolite used as an anti-obesity drug. The female plants are the major source of main isomer, (-) - hydroxycitric acid, a powerful inhibitor of citrate cleavage enzyme. G. indica being a polygamodioecious sp, differentiation between male and female plants is detected only at the flowering stage (approx. 7 yrs). Tissue culture methods, offer scope for large scale propagation of female plants. An efficient protocol for multiple shoots and callus has been standardized to produce HCA. Multiple shoots were obtained from immature seed explants on MS basal medium supplemented with NAA (2.69 mM), BAP (8.9 mM) and KN (0.93 mM). Elongation of shoots was achieved on half MS medium supplemented with NAA (0.54 mM), BAP (0.44 mM) and KN (0.93 mM). The shoots developed roots when they were treated with 4900 mM IBA for 30 seconds and cultured on half MS basal medium. Multiple shoots and callus were obtained from explants derived from in vitro developed plantlets. The regenerated plantlets were successfully transferred to soil in pots. Multiple shoots and the callus cultures produced HCA.

Keywords: Garcinia indica, organogenesis, micropropagation, hydroxycitric acid

 

 

 

Production of Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) in Callus and Cell Suspension Cultures of Plumbago indica Linn.

K Satheeshkumar and S Seeni

The relationship between growth and plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone) formation in callus and cell suspension cultures of Plumbago indica was investigated. Semi-friable callus was induced on young stem and leaf segments in MS solid medium containing 1.5-2.5 mgl-1 2, 4-D plus 0.5-1.5 mgl-1 KN. Cell suspension cultures were established from stem-derived semi-friable calli and cells released from aseptic leaf segments. Quantitative analysis of plumbagin in callus and cell cultures at different periods of growth revealed an increase in concentration of plumbagin with the increase in growth of cultures. Plumbagin level in both culture systems on unit dry wt basis was same throughout the growth of callus (0.05 mg/ g, dry wt) and cell cultures (0.028 mg/g, dry wt).

Keywords: Plumbago indica, callus, cell suspension cultures, plumbagin