Indian Journal of Biotechnology

 

Special Issue on
Plant Molecular Biology and Biotechnology

  

VOLUME 2

NUMBER 1

JANUARY 2003

 

CONTENTS

 

Hormonal regulation of moss protonema development and the possible origin of plant  hormonal responses in bryophytes

9

M M Johri & Jacinta S D’Souza

 

 

 

Hybrid necrosis in wheat–A genetic system showing reduced capacity to detoxify reactive oxygen species leading to programmed cell death

17

Geetanjali Sharma, B Srivalli & Renu Khanna-Chopra

 

 

 

Azolla-Anabaena symbiosis–From traditional agriculture to biotechnology

26

Anjuli Pabby, Radha Prasanna & P K Singh

 

 

 

Mutants for biochemical and molecular insights into embryogenesis in plants

38

Amita Bhattacharya, Preeti Sharma & Paramvir Singh Ahuja

 

 

 

Mass spectrometry: An essential tool for genome and proteome analysis

48

Pushpendra Kumar Gupta & Sachin Rustgi

 

Biotechnological importance of Piriformospora indica Verma et al–A novel symbiotic Mycorrhiza-like fungus: An overview

65

Anjana Singh, Archana Singh, Meera Kumari, Mahendra K Rai & Ajit Varma

 

 

 

Protoplasm fusion and brassica improvement

76

 P B Kirti, S Prakash, S R Bhat & V L Chopra

 

 

 

Molecular breeding for maize improvement: An overview

85

B M Prasanna & D Hoisington

 

 

 

Molecular diagnosis and application of DNA markers in the management of fungal and bacterial plant diseases

99

T R Sharma

 

Plant insecticidal proteins and their potential for developing tansgenics resistant to
insect pests

110

K R Koundal & P Rajendran

Molecular strategies for developing salt tolerant crops

121

Rajinder Kumar Jain, Navinder Saini, Sunita Jain & Randhir Singh

 

Agrobacterium-mediated transformation efficiency in blackgram and rice enhanced by multiple copies of pTiBo542 virB and virG

138

V Balaji, P Rajamuni, G Sridevi & K Veluthambi

 

Instructions to Contributors

147

 

 

 

 

Hormonal Regulation of Moss Protonema Development and the Possible Origin of Plant Hormonal Responses in Bryophytes

M M Johri* and Jacinta S D'Souza

Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India

 

The protonema of mosses is a far simpler paradigm to understand the mechanism of hormonal action and tolerance to abiotic stresses in plants. Its developmental biology, responses to hormones and the similarity of signaling mechanisms with higher plants are reviewed. There is strong evidence for second messenger role of calcium ions in the action of cytokinin. Multiple calcium-dependent protein kinases (CDPKs) are present in the protonema. The Funaria hygrometrica CDPK gene (FhCDPK) shows the characteristic catalytic and autoinhibitory domains, the four EF hands and the highest homology to CDPKs from higher plants but far lower to liverwort or other moss CDPK genes. A 38 kDa myelin basic protein kinase (MBP kinase) is activated within minutes by abscisic acid (ABA) and salinity. As ABA also confers tolerance against desiccation and freezing and the wheat ABA-inducible promoter is fully functional in mosses, the ABA signaling mechanism seems to be highly conserved. In plants, the CDPKs are involved not only in hormonal signaling but also in the acclimation response against abiotic stresses. The manipulation of signal transduction components such as transcription factors, CDPKs and calcineurin have emerged as viable strategies to genetically engineer the stress tolerant plants. There is increasing evidence to support the origin of plant hormonal responses at the level of bryophytes.

 

 

Hybrid Necrosis in Wheat¾A Genetic System Showing Reduced Capacity to Detoxify Reactive Oxygen Species Leading to Programmed Cell Death

Geetanjali Sharma, B Srivalli and Renu Khanna-Chopra*

Stress Physiology Laboratory, Water Technology Centre, Indian Agricultural Research Institute,  New Delhi 110 012, India

 

Hybrid necrosis in wheat is the premature gradual death of leaves and leaf sheath caused by two complementary genes Ne1 and Ne2 when brought together in a hybrid combination. Many promising wheat varieties are carriers of these genes, which limit the parental choice for transfer of desirable traits as the necrotic plants die without producing seeds. The leaves of Kalyansona ´ C306 and WL711 ´ C306 hybrids showed enhanced generation of superoxide radical and H2O2 than parents before and during progression of necrosis. Higher lipid peroxidation was an early event in hybrid necrosis and was accompanied by a loss in membrane permeability and cell viability. The anti-oxidant defense in necrotic hybrids was not well coordinated culminating in a persistent oxidative stress in the leaf especially in the chloroplast. The hybrid necrosis barrier was overcome in some crosses by culturing ears in medium containing antioxidants and F2 seeds have been obtained. Hybrid necrosis is a unique genetic system for studying the molecular mechanism of programmed cell death in plants. The cloning and characterization of Ne1 and Ne2 may provide a tool having wide applications in agriculture. The review compares hybrid necrosis with other PCD phenomenon in plants.

 

 

Azolla-Anabaena Symbiosis - from Traditional Agriculture to Biotechnology

Anjuli Pabby, Radha Prasanna and P. K. Singh*

National Centre for Conservation and Utilization of Blue-Green Algae, Indian Agricultural Research Institute,

New Delhi-110012

 

The Azolla – Anabaena symbiosis has attracted attention as a biofertilizer worldwide, especially in South East Asia. But its utilization and genetic improvement has been limited mainly due to problems associated with the isolation and characterization of cyanobionts and the relative sensitivity of the fern to extremes of temperature and light intensity. This paper reviews the historical background of Azolla, its metabolic capabilities and present day utilization in agriculture. An outline of biotechnological interventions, carried out in India and abroad, is also discussed for a better understanding of the symbiotic interactions, which can go a long way in further exploitation of this association in agriculture and environmental management.

 

 

Mutants for Biochemical and Molecular Insights into Embryogenesis in Plants**

Amita Bhattacharya, Preeti Sharma and Paramvir Singh Ahuja*

Institute of Himalayan Bioresource Technology, Palampur 176 061, India

 

Despite the advancements that have been made in understanding the intricate mechanisms of embryogenesis, a lot remains elusive. However, the understanding of the mutants of different stages of embryo development viz. tsII, gnom, mickey, lec etc. have opened up new vistas towards gaining insights into their biochemical and molecular basis of embryogenesis. Today, studies on mutants have not only played a major role in the easier manipulation of PGRs in vitro but have also led to the important discoveries of genes that are involved in the process of embryo pattern formation and in elucidating the underlying genetic, molecular and physiological mechanisms of inducing polarity, cotyledon initiation and pro-vascular tissue differentiation. The present review deals with the different mutants and the role they have played in the understanding of molecular and biochemical insights of somatic embryogenesis.

 

 

Mass Spectrometry: An Essential Tool for Genome and Proteome Analysis

Pushpendra Kumar Gupta* and Sachin Rustgi

Molecular Biology Laboratory, Department of Genetics and Plant Breeding, Ch. Charan Singh University,
Meerut 250 004, India

 

Mass spectrometry (MS), in its various forms, has become an essential tool for genome and proteome analysis. It involves gaseous ionization of the analyte to be examined, followed by separation of ions according to mass-to-charge (m/z) ratio and determination of molecular masses of ions from mass spectra obtained after mass spectrometry of analyte. Several methods for ionization, mainly including MALDI and ES, each coupled with a specific mass spectral analysis system (e.g. TOF-MS and quadrupole MS) are available. MS/MS is devised particularly for the determination of amino acid sequences of small peptide. The advantage of MS over other techniques is its speed, since gel electrophoresis and labeling of the analyte, needed in other techniques used for genome/proteome analysis, can be dispensed with. Applications of mass spectrometry for genome analysis include DNA sequencing and SNP detection, the latter involving PinPoint assay (minisequencing), PNA hybridization, invader cleavage, “MALDI on a chip”, etc. Similarly, its applications for proteome analysis include peptide sequencing, determination of molecular weights of proteins and protein identification by database search. Protein modifications and protein–protein interactions can also be examined by coupling mass spectrometry with database search. In this manner, mass spectrometry has become an essential tool for genome and proteome analysis.

 

 

Biotechnological Importance of Piriformospora indica Verma et al¾A Novel Symbiotic Mycorrhiza-like Fungus: An Overview

Anjana Singh1, Archana Singh2, Meera Kumari1, Mahendra K Rai3 and Ajit Varma1*

1 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

2Department of Biological Sciences, University of Alabama, Huntsville, AL 35899, USA

3 Department of Biotechnology, Amravati University, Amravati 444 602, India

 

Piriformospora indica Verma et al, a newly discovered root colonizing, AM fungi-like fungus, showed prominent positive influence on a wide range of plants of agriculture, forestry and flori-horticultural importance. Interestingly, P. indica has a wide host range of monocots and dicots including legumes, terrestrial orchids (Dactylorhiza maculata) and members of the bryophytes (Aneura pinguis). The fungus showed potential as an agent for biological control of disease against soil-borne root pathogens. 32P experiments suggest that this fungus is important for phosphorus acquisition by the roots, especially in the arid and semi-arid regions. Mycelium could utilize a wide variety of inorganic and organic phosphate chemicals and produced acid phosphatases at the tip of the hyphae. The fungus was found to act as an excellent tool for biological hardening of tissue culture raised plants (tool for biological hardening). Fungus can be axenically grown on a wide range of synthetic simple and complex media with sucrose or glucose as carbon energy source. Mass cultivation of the fungus can be easily achieved on simplified broth culture. The growth is best obtained between 25-35°C and pH 5.8. The fungus was discovered from the rhizospheric soils of desert plants, Prosopis chilensis Stuntz and Ziziphus nummularia Burm. f. in the sandy desert of Rajasthan, North-west India. For its characteristic spore structure the isolate was named Piriformospora indica. Electron microscopy revealed the presence of typical dolipore septum with continuous parenthosomes, which indicated that the fungus belongs to the Hymenomycetes (Basidiomycota). Sequences of 18S rRNA and 28S rRNA indicate that P. indica is related to the Rhizoctonia group and the family Sebacinaceae (Basidiomycetes). Immunofluorescence, ELISA, western blot and immuno-gold characterization indicated affinity of P. indica with the members of Glomeromycota, namely Glomerales, Diversisporales and Archeaosporales.

 

 

Protoplast Fusion and Brassica Improvement

P B Kirti1* and S Prakash2

1Department of Plant Sciences, University of Hyderabad, Hyderabad 500 046

2Formerly at the National Research Centre for Plant Biotechnology, Indian Agricultural Research Institute,
New Delhi 110 012

 

Brassica coenospecies has been bestowed with a large collection of wild and weedy relatives, which could act as donors of genes controlling agronomically important traits like disease resistance, yield attributes, modified fatty acid composition in the seed etc. Additionally, the cytoplasm of these species could be the sources for developing alloplasmic male sterility systems that are important in developing hybrid mustard varieties. The technology of protoplast culture and protoplast fusion has been very well developed for crop Brassicas and this could be effectively exploited in developing somatic hybrids and cytoplasmic hybrids, in short called as cybrids. This could pave the way for overcoming problems associated with the development of incompatibility barriers and allow for obtaining hybrids of the wild species with the cultivated species and manipulating characters that are controlled by organelle genomes. In Nicotiana species, protoplast fusion resulted in the development of novel mitochondrial genomes and plants carrying novel genomes have differing flower morphology. Protoplast fusion in Cruciferae also nearly invariably results in mitochondrial genome recombination/rearrangement. Hence, protoplast fusion can be effectively utilized in manipulating flower morphology and male sterility in crop Brassicas.

 

 

Molecular Breeding for Maize Improvement: An Overview

B M Prasanna1* and D Hoisington2

1Division of Genetics, Indian Agricultural Research Institute, New Delhi 110 012, India

2International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico, D F, Mexico

 

The maize genome is one of the most extensively analyzed among the plant  genomes. Consequently, maize has been at the forefront in development and evaluation of an array of molecular markers for varied purposes in genetics and breeding. Besides the well-demonstrated utility of molecular markers in genotype differentiation and analysis of genetic diversity in maize germplasm, application of DNA-based markers is also of considerable significance to tropical/sub-tropical maize production systems, such as in India, for mapping and marker-assisted selection for resistance to major biotic/abiotic stresses affecting production and productivity.  Significant impetus in this direction has been provided in recent years through the Asian Maize Biotechnology Network (AMBIONET). This article provides an overview of the recent efforts under AMBIONET in relation to: (i) the molecular characterization of inbred lines developed by various public sector institutions in India; (ii) the analysis of genetic diversity in the Indian maize germplasm using microsatellite markers; and (iii) the mapping of quantitative trait loci conferring resistance to different downy mildews affecting maize in tropical Asia. Judicious integration of conventional and molecular approaches in maize breeding programmes is vital for efficient utilization of genetic resources, and improving the production and post-harvest characteristics of the elite germplasm. This shall, in turn, require further strengthening of synergistic linkages and partnerships among national and international research institutions to harness the rapidly emerging information and technologies related to molecular breeding in maize.

 

 

Molecular Diagnosis and Application of DNA Markers in the Management of Fungal and Bacterial Plant Diseases

T R Sharma*

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

Successful management of plant diseases is mainly dependent on the accurate and efficient detection of plant pathogens, amount of genetic and pathogenic variability present in a pathogen population, development of disease resistant cultivars and deployment of effective resistance genes in different epidemiological regions. Beside conventional methods of pathogen detection and breeding resistant cultivars, recent developments in molecular biology techniques particularly the advent of various DNA markers have greatly influenced the plant protection methods. Pathogen detection has relied on isolation of microorganisms and observations of symptoms they induce on susceptible hosts. In many situations cultural, morphological and chemical markers have been used to study variation in pathogen populations. Such markers are limited, often unstable and are influenced by environmental conditions. Molecular detection of plant pathogens and characterization of genetic variability by using different DNA markers have offered additional tools in the hands of plant pathologists and plant breeders. Various PCR based and hybridization based DNA marker techniques can be used for the characterization of genetic variability in pathogens and molecular tagging of disease resistance genes. DNA markers linked to specific resistance gene can be used in marker-assisted-selection for resistance breeding, gene pyramiding and map-based cloning of the resistance genes. In this communication various uses of DNA markers in pathogen diagnostics and mapping, pyramiding and map-based cloning of disease resistance genes have been discussed.

 

 

Plant Insecticidal Proteins and their Potential for Developing
Transgenics Resistant to Insect Pests

K R Koundal* and P Rajendran

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

 

Insects cause heavy damage to cultivated crop plants. Production of proteinaceous inhibitors that interfere with the digestive biochemistry of insect pests is one of the naturally occurring defence mechanisms in plants. These proteins include lectins, arcelins and inhibitors of alpha amylases and proteases of various larvae pests. Use of plant genes encoding effective inhibitors of major digestive enzymes, such as protease and a-amylase inhibitors of the target pest species is emerging as viable approach for the production of pest resistant transgenic crop plants. Therefore, it is important to characterize proteins and their genes from our indigenous crops in order to strengthen and broaden our gene bank for pest control manipulations. The availability of diverse insecticidal proteins and their genes from different plant species will make it easier to use one or more genes in combination to develop resistant crop plants. Eventually, insect resistant transgenic plants will certainly prove more economic than any conventional control strategy if long-term benefit of transgenic crops especially factors such as environmental damage and human health risks are considered.

 

 

Molecular Strategies for Developing Salt Tolerant Crops

Rajinder Kumar Jain1, Navinder Saini1, Sunita Jain2 and Randhir Singh2*

1Department of Biotechnology and Molecular Biology, 2Department of Biochemistry,

CCS Haryana Agricultural University, Hisar 125 004, India

 

Salinity is one of the most important abiotic stresses for agricultural crops. High concentrations of salts cause hyperosmotic and ionic stresses, which, in turn, may generate secondary stresses such as oxidative stress, etc. The complexity and polygenic nature of salt tolerance trait has seriously limited the efforts to develop salt-tolerant crop varieties. This paper reviews new molecular strategies that have been or can be used for the molecular dissection of plant responses to salt stress, discovery of novel structural and regulatory genes involved in stress adaptation, and transgenic and molecular marker strategies used for engineering salt tolerance in plants. Application of novel techniques such as genome sequencing, high-throughput analysis of genomic-scale expressed sequence tags (ESTs), DNA chips/cDNA microarray analyses, targeted or random mutagenesis, knockouts, molecular mapping and gain-of-function or mutant complementation, is expected to accelerate the discovery of the new genes involved in stress adaptation as well as improve understanding of stress biology. A number of stress-related genes have been characterized including the ones that encode for important enzymes or a biochemical pathway, participate in signaling pathways or act as transcriptional regulators for coordinated regulation of stress related genes. Some of these genes have been successfully transferred in model plant species including Arabidopsis, rice and tobacco, and a marginal to significant improvement in salt-tolerance has been reported. In addition, molecular markers can be used for linkage mapping of genes/QTLs for salinity tolerance trait, marker-assisted transfer and pyramiding of such QTLs into agronomically desirable genotypes and/or for map-based cloning of genes. Application of transgenic and molecular marker research coupled with rapid gene discovery via functional genomic research in plants shall provide effective means for designing salt-tolerant crops.

 

Agrobacterium-mediated Transformation Efficiency in Blackgram and Rice Enhanced by Multiple Copies of pTiBo542 virB and virG

V Balaji, P Rajamuni, G Sridevi and K Veluthambi*

Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India

 

Broad host-range Inc W plasmids pBAL3 with 3' end of virB and complete virG of pTiBo542 and pToK47 with complete virB and virG were introduced into Agrobacterium tumefaciens vir helper strains harbouring a binary vector pBAL2 (Inc P). Transformation efficiencies of these strains were evaluated by transforming the primary leaf segments of blackgram [Vigna mungo (Linn.) Hepper cv. Co5] and the scutellum-derived calli of rice [Oryza sativa Linn. cv. Pusa Basmati 1]. Transformation was evaluated on the basis of GUS staining in stably transformed calli. In the octopine vir helper strain LBA4404 harbouring pBAL2, the plasmid pBAL3 with the 3' end of virB and complete virG of pTiBo542 increased transformation efficiency by 141% to 192%. The plasmid pToK47 that carried pTiBo542 virB in addition to virG did not promote transformation of blackgram any further. In the l, l-succinamopine vir helper strain EHA105, pBAL3 did not improve blackgram transformation. In rice, transformation of LBA4404(pBAL2) occurred only when multiple copies of pTiBo542 vir genes were present. Transformation efficiencies with pBAL3 and pToK47 were 10 and 18%, respectively. While, multiple copies of 3'end of virB and virG of pTiBo542 harboured on a compatible replicon were effective in blackgram, complete virB was additionally required for efficient transformation of rice.