eChapters

Objective: To check the integrity of isolated RNA on agarose gel.

Background information:

The most common method used to assess the integrity of total RNA is to run RNA sample on agarose gel (denaturing) stained with ethidium bromide (EtBr). While native (non-denaturing) gels can be used, the results can be difficult to interpret. The secondary structure of RNA alters its migration pattern in nativegels so that it will not migrate according to its true size. Nondenaturing conditions also result in bands that are not as sharp, and even multiple bands representing different structures of a single RNA species.

Intact total RNA run on a denaturing gel will have sharp, clear 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band. This 2:1 ratio (28S:18S) is a good indication that the RNA is completely intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit the 2:1 ratio of high quality RNA. Completely degraded RNA will appear as a smear (having very low mw). Inclusion of RNA size markers on the gel will determine the band size or smears size and will also serve as a good control for ensuring proper gel run. Note: Poly selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).

Objective: To isolate RNA from plants using TRIzol reagent and its quantification by spectrophotometer.

Background information

RNA (Ribonucleic acid) is a polymeric substance present in living cells and many viruses, consisting of a long single-stranded chain of phosphate and ribose units with the nitrogen bases adenine, guanine, cytosine, and uracil, which are bonded to the ribose sugar. RNA is necessary in all the steps of protein synthesis in all living cells and carries the genetic information for many viruses.

The isolation of RNA with high quality is a crucial step required to perform various molecular biology experiment. TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells and tissues (Chomczynski, 1993).TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components. Addition of chloroform, after the centrifugation, separates the solution into aqueous and organic phases. Aqueous phase contains RNA.

After transferring the aqueous phase, RNA can be recovered by precipitation with isopropyl alcohol. DNA and proteins can be recovered by sequential separation after the removal of aqueous phase. Ethanol precipitation needs DNA from the interphase, and an additional precipitation with isopropyl alcohol requires proteins from the organic phase. Total RNA extracted by TRIzol Reagent is free from the contamination of protein and DNA The isolated RNA can be further used in Northern blot analysis, in vitro translation, poly (A) selection, RNase protection assay and molecular cloning.

Objective:To transfer DNA on nylon membrane by Southern blotting.

Background information

A Southern blot is a method used in molecular biology to detect a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization.

The British biologist Edwin Southern (Southern, 1975) discovered this technique the technique is known by his name. Southern blotting technique uses digested genomic DNA with restriction enzymes. Southern blotting technique may be used to identify the gene copies number in a genome. A probe that hybridizes only to a single DNA segment that is uncut with restriction enzyme will produce a single band on a Southern blot, whereas multiple bands will likely be observed when the probe hybridizes to several highly similar sequences (e.g., may be due to sequence duplication).

In the southern blotting technique a short DNA probe is used that matches based on complementary DNA sequence fragment on filter membrane. It proves that the DNA fragment on membrane contain specific sequence for which DNA probe is prepared. When DNA is transferred on the membrane from gel it permits binding of radioactive/Biotin labeled hybridization probe to the restricted DNA. Southern blotting is also important for autoradiography.

Southern blotting is an important molecular biology technique that may be used for homology based cloning, screening a DNA library, screening of cloned DNA fragments and to identify methylated sites in genes.

Objective: To amplify DNA using RAPD primers and study on agarose gel.

Background information:

RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals. RAPD is applied to analyze the genetic diversity by random primers. Unlike traditional PCR analysis, RAPD does not require any prior DNA sequence information of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions complementary with primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the templ that was earlier complementary with primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel. RAPD is a dominant marker means it cannot distinguish between heterozygous; single copy and homozygous; two copies. Rarely RAPD markers may be Co-dominant where different fragment length of DNA is amplified from the single locus. PCR is a molecular technique used for amplification of DNA, a complete enzyme based chemical reaction, therefore, the concentration and quality of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible. Mismatches between the primer and the template may result in the total absence of PCR product or decreased amplified product. Thus, the RAPD results can be difficult to interpret.

Objective: To check quality of genomic DNA on agarose gel electrophoresis.

Background information

Agarose gel electrophoresis is a common method of gel based electrophoresis technique used in biochemistry, molecular biology, genetics and clinical chemistry for separation of Protein and nucleic acids from a mixture of macromolecules. Agarose is a major component of agar that is used as a matrix in gel baseelectrophoresis. The nucleic acids (DNA and RNA) are separated based on length of fragments while, proteins are separated based on size and/or charge (Kryndushkin, et al. 2003). For separation of bio-molecules the electric field is applied that leads to movement of charged particles via agarose gel matrix and bio-molecul s are separated based on difference in size in the agarose gel matrix (Sambrook and Russel, 2001).

Gel casting with agarose is easy and has very few charged groups, hence found most suitable for nucleic acid separation in most of the biochemistry, molecular biology and biotechnology laboratories. Separated DNA/RNA ma be seen and recorded with stain under UV light. Further DNA fragments can be eluted from agarose gel with an ease for further down applications. The percentage of agarose gel used is in the range of 0.7 - 2. Lower percent of agarose gel is used for long DNA fragments while, higher percent of agarose gel is used for short DNA fragments with suitable running buffer.

Objective: To isolate DNA from plant sample by CTAB method and quantification by spectrophotometer.

Background information:

DNA isolation from different plant tissues viz., leaf, stem, root, flower and seed is a challenging task due to hindrance of variety of variety of biochemical compounds present and further problem increases with diverge plant species. In case of animal tissues the diversity in cellular bio-molecules in different species is much lower compared to plants. Two major phyto bio-molecules that have wide variation between species and pose serious problems in DNA isolation are: polyphenols and polysaccharides. These phyto bio-molecules if contaminated in DNA then may hinder further down applications like PCR, restriction digestion, sequencing, etc.

Several methods have been developed and reported that can remove polyphenols and polysaccharides efficiently during DNA isolation from plants. For this purpose two different chemicals are widely used,the first one is CTAB (cetyl trimethyl ammonium bromide) that is a cationic detergent and helps in removal of polysaccharides and the second one is polyvinylpyrrolidone that helps in removal of polyphenols. Due to above properties the CTAB based DNA extraction buffer is very common for plant DNA extraction.

Significance of different types of chemicals found in CTAB based plant DNA extraction method is explained as under:

Objective: To study peroxidase isozyme variation in rice seedlings by electrophoresis.

Background information

For electrophoresis and PAGE please see practical no. 4. For peroxidase please see practical no. 7.(Page 17-22)

Isozymes (isoenzymes or more generally as multiple forms of enzymes) are the enzymes that are differing in the sequence of amino acids but they work on same substrate or in other words catalyze same biochemical reaction. The isozymes have difference in K_M values and regulatory properties. Isozymes are very useful for proper tuning of metabolic activities and to meet the specific need of particular tissue or developmental stage, e.g. lactate dehydrogenase (LDH). Isozymes are coded by homologous genes that have diversity during evolutionary process. Although, strictly speaking, allozymes represent enzymes from different alleles of the same gene, and isozymes represent enzymes from different genes that process or catalyse the same reaction, the two words are usually used interchangeably.

Objective: To extract and perform enzyme assay of Superoxide dismutase from plant.

Background information:

Superoxide dismutase (SOD, EC 1.15.1.1) is the enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide (O₂^-) radical into either ordinary molecular oxygen (O₂) or hydrogen peroxide (H₂O₂). Superoxide is produced as a by-product of oxygen metabolism and, if not regulated, may cause cell damage (Hayyan, et al. 2016). Hydrogen peroxide is also responsible for cell damage and may be degraded by other enzymes like catalase. Thus, SOD is an important antioxidant defense in nearly all living cells exposed to oxygen. One exception is Lactobacillus plantarum and related lactobacilli, which use a different mechanism to prevent damage from reactive O₂^-.

Objective: To extract and perform enzyme assay of guaiacol peroxidase from plant.

Background information:

Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:

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

Background information

Catalase is a commonly distributed enzyme and present in approximately all organisms exposed to oxygen (such as bacteria, plants and animals). Catalysis of H₂O₂ into H₂O and O₂ is done by catalase enzyme (Goodsell, 2004).

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).

Carbon is found in all living organisms and is the major building block for life on earth. It exists in many forms, predominantly as plant biomass, soil organic matter, and as the gas CO₂ in the atmosphere and dissolved in sea water. Carbon cycles globally among five distinct pools among which the largest oceanic poolis estimated at 38000 Pg (Pg= petagram =1x10¹⁵g=billion tonnes) and is increasingat the rate of 2.3 Pg C yr^-1 (Fig. 1). The geological C pool, comprising fossil fuels, is estimated at 4130 Pg, pedologic, estimated at 2500 Pg to 1m depth, atmospheric pool comprising 760 Pg of C with nearly all of it as CO₂, and the smallest among theglobal C pools is the biotic pool estimated at 560 Pg. The pedologic and biotic C pools together are called the terrestrial C pool estimated at approximately 2860 Pg.

Agricultural systems are complex, and understanding this complexity requires systematic research under shrinking agricultural research resources. Field experimentation can only be used to investigate a very limited number of variables under a few site-specific conditions. The crop growth simulation models simulate crop growth, development and yield taking into account the effects of weather, genetics, soil water, carbon and nitrogen, planting, irrigation and fertilizer (e.g. Nitrogen, Phosphorus) management. Ofthe many factors that potentially affect the growth of plants, the two that are most responsive to management are water and nitrogen. It is therefore not surprising that most crop models include routines to handle responses to water and nitrogen. Less frequently do models attempt to describe limitations due to other soil constraints (Probert and Keating, 2000). In the context of integrated nutrient management, the performance of a crop model depends mainly on its ability to adequately describe the release of nutrients from diverse inputs and their uptake by the crop.

Origins and genesis of a crop simulation model

Crop simulation model approach was started near about 50 years ago, in early 1960s when early simple crop models were developed for estimation of transpiration and photosynthesis. The first step towards crop modeling was the development of simple models to estimate light interception and photosynthesis (Loomis and williams, 1963; De wit, 1965 and Duncan et al., 1967). There are many crop growth simulation models. Some are more generic in nature while others are built for specific purposes. Most of these models simulate crop growth and soil processes using daily time steps. All models are developed with some assumptions and hypotheses, and all have strengths, weaknesses and limitations for appropriate application. Well-known crop modelling groups across the world include IBSNAT/IFDC (International Benchmark Sites Network for Agro technology Transfer/International Fertilizer Development Centre) in USA, WAU/AB-DLO (Wageningen Agriculture University/Centre for Agro-biological Research) in the Netherlands, and APSRU (Agriculture Production Systems Research Unit) in Australia. The IBSNAT project was initiated in 1982, and over the past 20 years it has developed more than 15 models, including CERES (Crop Estimation through Resource and Environment Synthesis) Rice and CERES Wheat, which are available either as stand-alone models or within the DSSAT (Decision Support System for Agrotechnology Transfer) shell (Uehara and Tsuji, 1993).

Introduction

Seed spices include group of annuals whose dried fruit or seeds are used as spices. These are characterised by pungency, strong odour, sweet or bitter taste. Coriander, cumin, fennel and fenugreek occupy largest area among the seed spices. However, ajowan, dil, celery, anise, nigella and caraway having secondary place as far as area and production is concerned. Major area under seed spices is in Rajasthan and Gujarat, and other seed spices producing states are Chattisgarh, Madhya Pradesh, Andhra Pradesh, Tamil Nadu and Uttar Pradesh. The seed spices account for about 36% and 17% of the total area and production of spices in country. The export of seed spices from India during 2008-09 was 4.71 thousand tonnes worth Rs. 54914.8 million rupees. The per cent export growth rate during 1995-96, 2000-01, 2005-06, 2007-08 and 2008-09 was 12.0, 11.0, 8.0, 36.0 and 19.0% in terms of value and 13.0, 3.0, 8, 13.0 and 6.0 in respect of quantity, respectively.

Integrated plant nutrient management includes use of organic manure/ compost, bio-fertilizer, chemical fertilizer, green manuring, residue management, legume based cropping system, use of nutrient-responsive varieties, proper method and time oforganic manure and fertilizer application, soil and water management to minimize the nutrient losses occurring through volatilization, denitrification, runoff and leaching. Application of plant nutrients in proper balance form is also a part of Integrated Plant Nutrient Management (IPNS) system. Supply of nutrients to seed spices in appropriate quantities and at the correct times is essential for economically and environmentally sustainable agriculture. Soil organic matter, crop residues and manures play a vital role in the supply of macro and micronutrients and the transformation between the various organic and inorganic forms often control availability, both for plant uptake and loss to the environment (Aishwath and Vashishtha, 2008; Lal, et al., 2009).

Cost is the value of the factors of production used in producing and distributing goods and services. The cost of a factor unit equals the maximum amount which the factor could earn in alternative employment. Broadly speaking, cost is the measure of what has to be given up in order to achieve something. Production costs play an important role in the decision of the farmers. Cost of production here means the exogenesis incurred per unit of output. In general, at given level of prices, a farmer can increase his farm income in two ways i.e. (i) by increasing production or (ii) by reducing the cost of production. The cost of production involves both cash cost and non cash cost items. Cash costs are incurred when resources are purchased and used immediately in the production process such as casual labour, fertilizer fuel etc. And non cash cost like depreciation on farm building and equipment and payment made to farmer himself or family labour, management and owned capital. Before estimating the cost of cultivation/production, we should know the concepts/terms are used in estimating the cost. Concept means idea underlying or general notion.

Cost concepts and items of cost

The cost of production of a crop is considered at three different levels viz., cost A, cost B and cost C. The concept of nine costs such as is followed by the Directorate of Economics and Statistics, Ministry of Agricultures Government of india in their cost studies are given below.

Balanced crop nutrition is a most reaJized critical factor in allowing crops to give their full yield potential. The application of macro and micronutrients through manures and fertilizers to achieve this balance is an integral practice in modern crop production system. Macro and microelements have long been recognized as being associated with changes in the level of many diseases and yield and /or quality of crops. Three major elements N, P, and K are the most studied nutrients which chiefly affect relative resistance of crop plants to various diseases. Others macro/micro/beneficial nutrients including Ca, Mg, S, Mn,Te, Zn, Mo, Si, Bo and Cu have shown varying degrees of disease suppression/induction on many crop species under various environmental conditions. Crop nutrition influence plant health through: i. affecting the growth, reproduction, virulence, and survival of pathogens; ii. resistance and tolerance, predisposition and escape in the host crop; and iii. root exudates and microbial interactions facilitatingbiological control. Balanced crop nutrition can protect crops from pathogens by: i. avoiding plant stress, which may allow crops to better withstand the attack of pathogens and ii. manipulating nutrients to the advantage of crops and disadvantage of the pathogen. The modern intensive agriculture which mainly relies on chemical inputs (fertilizers and pesticides) and fertilizer responsive HYVs (high yielding varieties) has led the self sufficiency in food production in India and several other countries of the world. But, the indiscriminate and imbalance use of chemical fertilizers along with exhaustive cropping initiated several second generation problems including widespread micronutrient deficiencies and increased attack of several insect-pests and pathogenic diseases. The excessive use of nitrogen and no or reduced application of phosphorus, potash and other micronutrients is creating conducive conditions for several crop pathogens and their insect vectors. This situation frequently results in serious crop losses during favourable environmental conditions for disease development. The specific role of macro and micronutrients in maintaining crop health, effect of their dose and form of application on suppression /induction of various diseases and interaction with other soil/ environmental factor (s) will be discussed in following sections.