eChapters

Objective: To extract and perform enzyme assay of polyphenol oxidase from plant.

Background information:

Polyphenol oxidase (PPO or monophenol monooxygenase or polyphenol oxidase I, chloroplastic) is a tetramer that contains four atoms of copper per molecule, and binding sites for two aromatic compounds and oxygen (www.worthington-biochem.com). The enzyme catalyses the o-hydroxylation of monophenol molecules in which the benzene ring contains a single hydroxyl substituent to o-diphenols (phenol molecules containing two hydroxyl substituents). It can also further catalyse the oxidation of o-diphenols to produce o-quinones.

PPO causes the rapid polymerization of o-quinones to produce black, brown or red pigments (polyphenols) that cause fruit browning. The amino acid tyrosine contains a single phenolic ring that may be oxidised by the action of PPOs to form o-quinone. Hence, PPOs are some time referred as tyrosinases (Mayer, 2006).

Jolley et al. (1974) referred to PPO as 4 electron-transferring phenol oxidase and oxygen. It is responsible for browning reactions throughout the phylogenetic scale.

Objective: To separate protein fragments by using SDS-PAGE method.

Background information

Electrophoresis is the motion of dispersed particles relative to a fluid under the effect of limited uniform field of electricity (Dukhin, and Derjaguin, 1974). Ferdin and Frederic Reuss (Reuss, 1809) first time observed such phenomena of electrokinetic nature in 1807. They found that the clay particles move in the water by giving constant supply of current. It happened due to interface of charged particles surface and the surrounding media. The technique is used widely in chemistry, biochemistry and molecular biology to separate different molecules based on difference in charge, binding affinity and size. Electrophoresis may be of two types viz., Anaphoresis and Cataphoresis. Anaphoresis is the electrophoresis of anions (-ve charged ions) while, Cataphoresis is electrophoresis of cations (+ve charged ions). Electrophoresis is a major analytical technique in biochemistry that is used for separation of macromolecules like protein, enzymes and nucleic acids. Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size.

PAGE is abbreviated for Polyacrylamide gel electrophoresis that describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule.

Objective: To estimate total protein in the plant sample by Folin-Lowry's method.

Background information:

Protein assay can be done to determine the level of protein in solution by the Folin-Lowry biochemical method. The concentration of protein leads to change in color of the solution and based on different color in different protein concentration solution the change in color may be digitized by colorimeter or spectrophotometer. This technique was developed by Oliver H. Lowry in the year 1940s. The tyrosine and trytophan amino acid residues in a protein sample produces purple or blue color in the absorption maxima at 660 nm by using Folin-Ciocalteau (having phosphate and sodium tungstate molybdate) chemical reagent. The color intensity of protein sample depends on the concentration of above aromatic amino acids (tyrosine and tryptophan), hence every sample will vary for color. For protein estimation the BSA (Bovine serum albumin) is widely used as standard protein molecule due to its cost effectiveness, purity and easy availability. This method of protein estimation is very sensitive and can detect up to 10 μg/ml. This is a relative method of protein estimation and may be interfere by salts, Tris buffer, nonionic & cationic detergents, EDTA, lipids and carbohydrates. For reproducible assay the incubation time, pH (9-10.5) is critical in this method (Lowry, et al. 1951).

Objective: To identify salt tolerant genotypes by Glycine Betaine (GB) method in laboratory.

Background information:

Salt or salinity is a biggest danger that causes reduction in crop productivity worldwide. Salinity reduces the germination percentage, plant vigour and ultimately loss in final crop yield. There are different physiological parameters that are affected by high level of salinity viz., oxidative stress, physical alteration of bio-membrane, nutritional imbalance, loss of cell division & cell expansion, water stress, genotoxicity and ion toxicity. Together, these effects reduce plant growth, development and survival. During the onset and development of salt stress within a plant, all the major processes such as photosynthesis, protein synthesis and energy and lipid metabolism are affected. During initial exposure to salinity, plants experience water stress, which in turn reduces leaf expansion (Petronia, et al. 2011).

The water-soluble compounds which, are low in molecular weight, accumulate at certain concentration known as “compatible solutes” or “osmolytes” is the common strategy adopted by many organisms to combat the abiotic stresses. Some common compatible solutes are betaines, sugars (mannitol, sorbitol and trehalose), polyols, polyamines and amino acid (proline). Their accumulation is favored under water-deficit or salt stress as they provide stress tolerance to cell without interfering cellular machinery. The tolerant or sensitive species show difference in the stress tolerance level based on levels of accumulation of these compounds during abiotic stress condition. Accumulation of osmolytes like glycinebetaine (GB) in cells is known to protect organisms against abiotic stresses via osmoregulation or osmoprotection. Other roles of GB like cellular macromolecule protection and ROS detoxification have been suggested as mechanisms responsible for abiotic stress tolerance. In addition, GB also influences expression of several endogenous genes in plants.

Objective: To identify drought tolerant genotypes by PEG method in laboratory.

Background information:

Among different abiotic stresses, drought is considered to be most detrimental for agricultural production. A drought is a period of below-average precipitation in a given climatic region that results in prolonged shortages in water availability, whether atmospheric, surface water or ground water. A drought can last for several months or years. Drought has substantial impact on ecosystem and agricultural productivity.

Some plant species are better able to tolerate drought stress than others. Genetic variability for drought tolerance also exists within many plant species (Blum, et al. 1980). The efficient exploitation of that variability for crop improvement would be facilitated by a fast and accurate screening method for identifying drought tolerant genotypes (Clark and Mc Craig, 1982). Several methods utilizing seedlings to screen for drought tolerance have been developed using an osmoticum such as polyethylene glycol (PEG) (Singh, et al. 1984). The other synonyms of PEG are Poly ethylene oxide (PEO) and Poly oxyethylene (POE); these other chemicals depend on the molecular weight. Chemical structure of PEG may be written as H”(O”CH₂”CH₂)_n”OH (Kahovec, et al. 2002). PEG is used in certain abiotic stress experiments to produce high degree of osmotic pressure, normally in the order of 'tens of atmosphere' PEG produces osmotic pressure due to its flexible nature and water soluble polymer. PEG also makes certain specific interactions with bio-molecules. Due to above properties PEG is used as a specific chemical to produce osmotic pressure in several biochemical and bio-membrane related experiments. For some genotypes, the seedling response apparently is a good indicator of the plant's reaction to drought stress later in its life cycle. However, early screening of seed plants via seedlings tests must still be considered as a method for identifying genotypes that will tolerate drought stress in the vegetative stage (Andersen, et al. 1987).

Accorinti, J.1983. Antifungal products from algal origin. Rev Int Ocean Med, 72, 45-53.
Allnutt, F.C.T. 1996. Cyanobacteria (Blue -Green Algae) Biotechnology: Past, Present and Future. Journal of Scientific and Industrial Research. 55, 693-714.
Ananthanarayan, R. and Paniker, C.K.J. 2005. Textbook of Microbiology (VII Ed.) Orient Longman Pvt Ltd, Chennai-600 002. pp 1-657.
Asthana, R.K., Srivastava, A., Singh, A.P., Deepali, Singh, S.P., Nath, G., Srivastava, R. and Srivastava, B.S. 2006. Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree. Journal of Applied Phycology. 18, Number-1, 33-39.
Ayehunie, S., Belay, A., Baba, T.W., and Ruprecht, R.M. 1998. Inhibition of HIV-1 replication by an aqueous extract of Spirulina platensis (Arthrospira platensis). J. Acquir. Immune. Defic. Syndr. Hum. Retrovirol. 18, 7-12.

The objective of the present investigation was to evaluate the antibacterial properties of cyanobacterial species viz., Anabaena variabilis, Anabaena fertilissima, Nostoc muscorum, Nostoc punctiforme, Nostoc linckia, Nostoc commune, Spirulina platensis., Westiellopsis prolifica and Hapalosiphon sp. against some pathogenic species including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pnumoniae and Salmonella typhi. The following are the major findings of the study.

    •    Extracts prepared in methanol were found more active as compared to ethyl acetate, hexane and dichloromethane.
 
    •    Antibacterial activity of extracts varied with the test organisms. Maximum antibacterial activity was shown against Staphylococcus aureus followed by Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi. Klebsiella pneumoniae was found to be least susceptible against all tested cyanobacterial extracts.

    •    Antibacterial activity was observed against both Gram-positive and a spectrum of Gram-negative bacteria by almost all the cyanobacterial species examined in the present study except Westiellopsis prolifica where activity was observed only against S. aureus, a Gram-positive bacterium.

Present work was aimed to determine the antibacterial activity and minimum inhibitory concentration (MIC) of active crude extracts of selected cyanobacterial species. Nine cyanobacterial strains viz. Anabaena variabilis, A. fertilissima, Nostoc muscorum, N. punctiforme, N. linckia, N. commune, Spirulina platensis, Westiellopsis prolifica and Hapalosiphon sp. belonging to family; Nostocaceae, Oscillatoriaceae and Stigonemataceae were selected from the herbarium of Indian Agricultural Research Institute, New Delhi, and maintained in BG-11 growth medium throughout the study in the laboratory.

In the present investigation, nine cyanobacterial species viz. Anabaena variabilis, Anabaena fertilissima, Nostoc muscorum, Nostoc punctiforme, Nostoc linckia, Nostoc commune, Spirulina platensis, Westiellopsis prolifica and Hapalosiphon sp. were tested for their activity pattern against some selected bacterial species.
 
Each cyanobacterial species was initially subjected to determine the early stationary phase of their life-cycle, which is believed to be the most appropriate period for the production of secondary metabolites by an organism. The growth curve for each cyanobacterial species was prepared by culturing them in BG-11 growth medium. Optical densities (OD) recorded at 650 nm were plotted against number of incubation days for each cyanobacterium. After 23-25 days of incubation period, each culture was harvested to obtain the biomass and spent medium, which were then subjected to process of extraction by using various solvents viz., Hexane, ethyl acetate, dichloromethane and methanol. The extracts and spent medium thus obtained were then investigated for antibacterial activity against five pathogenic bacteria including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pnumoniae and Salmonella typhi.

Nine cyanobacterial strains viz., Anabaena variabilis, A. fertilissima, Nostoc muscorum, N. punctiforme, N. linckia, N. commune, Spirulina platensis, Westiellopsis prolifica and Hapalosiphon sp. were cultured in the laboratory. The growth curve for each culture was studied by growing them in Erlenmeyer flask using BG-11 growth medium. They were examined to determine the early stationary phases of their life-cycle, which are believed to be the most appropriate period for the production of secondary metabolites by an organism. After 23-25 days of incubation period, each culture was harvested and spent medium was collected by filtration of the biomass. Hexane, ethyl acetate, dichloromethane and methanol were used as the solvents for obtaining the extracts from biomass and spent medium of each culture which were then investigated against five pathogenic bacteria viz., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella typhi. Antibacterial activity of different cyanobacterial sp. has been shown in various tables.

Culture Media and Reagents

The media used throughout the study were procured from Hi- Media Laboratories, Mumbai, India and the chemicals used were procured locally. Composition of each medium is as follows:

Cyanobacterial species

Cyanobacterial species viz., Anabaena variabilis, Anabaena fertilissima, Nostoc muscorum, N. punctiforme, N. linckia, N. commune, Spirulina platensis, Westiellopsis prolifica and Hapalosiphon sp. were obtained from National Center for Conservation and Utilization of Blue-green Algae, Indian Agricultural Research Institute, New Delhi. Cultures were maintained in BG-11 growth medium throughout the study in the laboratory.

Cyanobacteria have been identified as one of the most promising groups of organisms from which novel and biochemically active natural products are isolated. Cyanobacteria such as Microcystis, Anabaena, Nostoc and Oscillatoria produce a great variety of secondary metabolites. The only group comparable to cyanobacteria is actinomycetes, which has yielded a tremendous number of secondary metabolites. The rate of discovery from traditional microbial drug producer like actinomycetes, which is in the focus of pharmaceutical research for decades, is decreasing and it is the time to turn to cyanobacteria and exploit their potential. Recently, there has been an increasing interest in cyanobacteria as a potential source of new drugs (Glombitza and Koch, 1989; Schwartz et al., 1990). This is of paramount importance to fight increasingly resistant pathogens and newly emergent diseases (Hayashi et al., 1994).

Cyanobacteria are photosynthetic microorganisms that have flourished on earth for more than 3 billion years with some genera showing only minor morphological changes since that time (Hagmann and Juttner, 1996). The medicinal and nutrient qualities of cyanobacteria were first appreciated as early as 1500 BC, when Nostoc species were used to treat gout, fistula and several forms of cancer (Pietra, 1990; Bladon, 2002; Liu and Chen, 2003).

Some cyanobacteria (Blue-green algae) like Nostoc are known to work as one of the two coponenents (photosynthetic partner or phycoboint) of lichens. The second partner of lichen is a lichen fungus or mycobiont. The lichens have not been described as a therapeutic agent in Charak Samhita, the monumental work on materia medica prepared by great Acharya Charak and his group in India more than three thousand years ago. However, these have been used in folklore medicine in many countries of the world and have been regarded to bear antibacterial properties (Katiyar and Katiyar, 2009). They are found in tundra even, the treeless plain of the arctic regions of Russia, Siberia, marshy in summer and frozen hard in winter. It is common to see gunny bags full of lichens for sale in the Masala market near Kesa House, Express Canal Road, at Kanpur. They also comprise a part of meat masala (spices for meat). There are references that Raja (King) Todarmal studied algae in relation to purification of water.

However, the search for antibiotics began in the late 1800s, with the growing acceptance of the germ theory of diseases; a theory which linked bacteria and other microbes to the causation of a variety of ailments. As a result, scientists began to devote time searching for drugs that would kill these disease-causing bacteria.

Introduction

Bioremediation can be defined as elimination, attenuation or transformation of polluting or contaminating substances by the use of biological processes, to minimize the risk to human health and the environment. Bioremediation of contaminated soil and water uses microbes (e.g. bacteria and fungi) to convert pollutants to harmless or more environmentally acceptable products and minimizes the risk to human health in a natural or managed process.

Introduction

Soil forms the base of the lithosphere on earth and is a fundamental and irreplaceable versatile three dimensional dynamic system that supports majority of terrestrial plant and animal life on earth. This soil system contains mineral (sand, slit and clay forming basic soil structure) and organic matter as well as marginal air and water contents. The soil organic matter content of the soil system constitutes every fraction of matter in various stages of decomposition that once was associated with the body, body parts, exudates, tissues and cells of living organisms including plants, animals and microorganisms. It is the product of continuous cycling of the compounds synthesized by autotrophic plants among the three kingdoms viz microbes, plants and animals. The decomposition of the organic residues in soil is performed primarily by the resident soil microflora termed as microbial decomposers; though initially the larger and complex organic material is broken down, devoured upon, chewed, regurgitated and transformed into smaller bits by larger soil faunal forms (earthworms, beetles, springtails, and collembola, the macro (large) to mesofauna) on which soil microbial forms could act upon.

Introduction

Introduction of improved high yielding varieties of crop plants, use of modern agricultural practices coupled with essential control of pests of food and fiber crops have significantly contributed towards increasing the food production in green revolution era in India. Pesticide application forms the foremost limb in hiked agricultural food production and management practices. The production and post production management of crop yield requires increased use of pesticides.

Introduction

India being a developing nation is trying to reach a balance in ever increasing population with marginal increase in crop production. There occurred a bigbang in crop production during the green revolution era however this also resulted in creation of infertile, barren lands with low chemical nutrient input responsive soils. Soil fertility is the major constraint among resource poor Indian farmers as lesser benefits are harnessed by use of newer agricultural protocols/tools/products viz improved varieties, productive technology from less fertile soils. There is dire need for looking for the alternative technologies of food or crop production to satiate the hunger of burgeoning Indian population. Today there is enormous emphasis on the conservation facets of soil fertility and crop productivity which are the instrumental philosophies in sustainable agriculture. Biofertilizers are the cost effective, ecofriendly and renewable resource of plant nutrients that could well supplement the use of already functional and popular chemical fertilizers for obtaining increase in crop yield without over hazardous impacts on various facets of soil chemistry, fertility and microbial diversity to attain sustainable agricultural system.

Introduction

Vesicular arbuscular mycorrhizae are fungi that exhibit symbiosis with roots of higher plants. The term vesicular arbuscular is derived from characteristic structure i.e. ‘branched hyphae or arbuscles’ that are formed with in the root cortical cells and vesicles or ‘swollen lipid storing organs’ that occur with in or between them.

Introduction

Lithosphere, including the earth’s crust, is helpful in providing the base or medium for sustenance of most life processes and forms, interwoven in fine network interdependencies within themselves and the environment surrounding them i.e. ecosystems. Terrestrial ecosystem contains an important dynamic living matrix termed soil, which is critical for agricultural production and food security. Soil forms the medium for growth and maintenance of a variety of plant species while the plant roots in the soil represent a four dimensional region in space and time of profuse activity relative to the bulk soil, revolving around pH, nutrient, redox potential, and exudate gradients changing as distance from the root increases (Marschner, 1995). The soil region adhering the plant roots is being strongly influenced by the presence of these roots and is characterized by high rates of microbial population and activity and referred as rhizosphere (the soil adhering to plant roots).