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Preface It is the valid experimentation that generates universal hypothesis in science. Theoretical information undoubtedly originates from practical and therefore a scientist through in modern practical orientation can be a scientist of high academic accomplishment. It is the valid experimentation that generates universal hypothesis in science. Theoretical information undoubtedly originates from practical and therefore a scientist through in modern practical orientation can be a scientist of high academic accomplishment. Our attempts are to make both Post Graduate student as well as teachers familiar with such captive world.We do not claimcurrent furnishings of techniques in any way exhaustive, but we do hope that it may certainly be to value to our readers. Techniques related to various physiological phenomenon are subject of tremendous interest and importance to plant physiologist, agronomist, horticulturist, ecologist, and biochemists. This book is intended to provide recognised methods related various plant processes in a comprehensive form. Text begins with what a researcher is primarily supposed to know before he actually goes for estimations. Further, you may see techniques on crop physiology such as hydroponics and plant nutrition, test for various stresses, water potential and water flow in plants, canopy gas measurements (Photosynthesis, Respiration andTranspiration), basic equations for growth studies and methods for estimations of plant products, microclimate. Efforts were also made to incorporate the topic like Climate Change and theory of phytotron as well as rhizotron in this book. In this book research worker must be familiar with latest procedure to elucidate the problems. The validity of the results based on fundamentals principles of physics. This book is meant to be used in conjunction with a standard text of plant physiology though elementary principles relating to the techniques are briefed. The subjects on hormones, tissue culture and seed technology are useful for students. Hope this book shall serve the need of students, teachers and researchers. The suggestions and advice are most welcome for the improvements in structural and for additions of current techniques. We shall feel successful in our efforts publishing this book if we know how this text has become meaningful to our readers in planning their research strategies. We would appreciate suggestions from teachers and students who use the book. Hope this book shall serve the need of students, teachers and researchers. The suggestions and advice are most welcome for the improvements in structural and for additions of current techniques. We shall feel successful in our efforts publishing this book if we know how this text has become meaningful to our readers in planning their research strategies. We would appreciate suggestions from teachers and students who use the book on how we could improve upon it.

Crop physiology is the study of the ways in which plant physiological processes are integrated to cause whole plant responses in communities. The subject matter of crop physiology includes the ways in which the knowledge of plant physiology is applied for better management of crops. Genetic potential of a plant and its interaction with environmental factors decides its growth and development by influencing or modifying certain internal processes. Plant physiology studies about these internal processes and their functional aspects. It helps to understand various biological processes of the plants like photosynthesis, respiration, transpiration, translocation, nutrient uptake, plant growth regulation through hormones and such other processes which have profound impact on crop yield. Many aspects of Agriculture can benefit from more intensive research in plant physiology to provide practical solutions in agriculture. Understanding the physiological aspects of seed germination, seedling growth, crop establishment, vegetative development, flowering, fruit and seed setting and crop maturity provides a reasonable scientific base for effective monitoring and beneficial manipulation of these phenomenon’s. Studying this phenomenon with a view to develop better crop management practices forms the subject matter of crop physiology. Later on several research works were carried out to understand the processes like translocation of food materials, their partitioning towards economic yield, storage mechanisms, physiology of flowering, effect of stressful environmental factors on crop growth and development, role of plant growth regulators in increasing the crop productivity etc. All these areas have enriched the knowledge of physiological processes and their role in deciding the crop yield. Crop physiology covers different topics such as seed germination and proper establishment of seedling depends upon various internal and external factors. Knowledge in seed physiology helps in understanding of different physiological and morphological changes that occur during germination. Any deviation in these processes causes seed dormancy. The dormant condition of the seed, bars immediate use of harvested seed for next crop which is important in intensive agriculture. By understanding the causes and effects of this problem, crop physiologists have come up with different methods of breaking the seed dormancy. Nutriophysiology is yet another important area to understand crop physiology. For the healthy growth of a crop, around 16 essential elements are required. Knowledge of nutriophysiology has helped in identification of essential nutrients, ion uptake mechanisms, their deficiency symptoms and corrective measures. It also helps to check the toxicity symptoms of various nutrients.

Basic LabTechniques - This laboratory is intended to: (1) Provide an introduction to basic techniques used in biology labs (2) provide some experience with the equipment employed in future labs 2.1 Microscopy The compound light microscope is a veryimportant tool in the study of biology. Typical compound light microscopes allow specimens to be viewed at 400 to 1000 times their actual size (total magnification is determined by multiplying the objective lens magnification by the ocularlens magnification). Thelight microscopeis made up of a system of lenses aligned in such a way thatallows magnification, as well as improved resolution. The resolution obtainable is also dependent on contrastin the specimen. Contrast can be adjusted with the iris diaphragm, located below the condenser lens,and can also be enhanced by the addition of stains to the specimen. Scientific drawings of specimens observed under the light microscope are often used to provide a record of what was viewed. Proper scientific drawings should be neatly drawn,with smooth lines, in pencil (they should never be in colour). More than one cellshould be drawn to illustrate the cellularassociation to the observer(e.g.found singly or as part of a tissue). Labels should be added using straight horizontal lines to the right side of the drawing, and care should be taken with singular and plural forms of structural terminology. To demonstrate differences in density of specific structures, use stippling rather than shading. A figure caption thatindicates the specimen viewed, how viewed, and the to right side of the drawing, and care should be taken with singular and plural forms of structural terminology. To demonstratedifferences in densityof specific structures, use stippling rather than shading. Afigure caption thatindicatesthe specimen viewed, how viewed, and the magnification of the drawing is always necessary.

A metabolic cell contains about 75-95% water by weight. It is important for plants for proper growth and development. Any significant reduction in availability will affects metabolic activity and thus growth parameters which determine yield. The uptake of water by the plant (roots) from the soil is an osmotic phenomenon. The water potential represents the free energy per moles of water. It can be reduced by: Decreasing temperature Adding soluble pressure or Decreasing pressure or Providing additional absorptive surface

Objective: To estimate qualitatively various chlorophyll fractions Materials Required: Leaf material, acetone, petroleum ether, distilled water, methyl alcohol, potassium hydroxide, diethyl ether, separating funnels, stand beakers and conical flasks. Principle: In biological systems the molecule absorbing radiant energy in the visible range is referred as a pigment. To the human eye these pigments are coloured: chlorophyll is green, carotenoids are red or yellow, xanthophylls is yellow and phytochrone is blue. These pigments are complex molecules. Chlorophylls are the most important pigments active in photosynthesis. At least nine different types of chlorophylls are known – chlorophyll a, b, c, d, e bacteriochlorophylls a and b and chlorobium chlorophylls 650 and 660. Chl a and chl b are best known pigments. In addition to chlorophyll, carotenoids, xanthophylls etc. Are also present as accessory pigments. The chlorophylls are directly involved in photosynthetic electron transport the other pigments serve as accessory pigments and they also serve the purpose of light harvesting. There are different organic molecules are soluble in different organic solutions, however, their solubility is differential in different chemicals. Based on the nature the chlorophyll pigments can be separated and identified.

Stomata are pores formed between two specialized epidermal cells, the guard cells, which are found on the surface of aerial parts of higher plants (leaves). They control the gas exchange between the leaf and the ambient air. Their main functions is to facilitate the entry of CO2 into the leaf for photosynthesis and loss of water vapour for evaporative cooling of the leaves. The guard cells are formed by the transverse division of stomatal mother cells that differ morphologically and biochemically from other epidermal cells. The middle lamella disintegrates and the two walls separate leaving an opening between them the stomatal pore. The walls bordering the pore become considerably thickened. They are generally cutinised. This radial thickening of the inner wall plays an important role in stomatal movement. Stomata control 95 per cent or more of CO2 and water vapour exchange between the leaf and atmosphere. Location and occurrence of stomata: Stomata are found in all higher plants down to the evolutionary level of the phylum Bryophyta. Stomata are not found in liverworts. In the leaves of most herbaceous plants the stomata are found in both upper (Adaxial) and the lower (Abaxial) surfaces. Such leaves are known as Amphistomatus. Usually in tree species, stomata are found only on the lower surface of the leaves. Such leaves are called Hypostomatus. Aquatic plants with floating leaves such as water lilies have stomata only on the upper surface and such leaves are called Epistomatus. Most of submerged aquatic plants do not posses stomata and diffusion of gases occurs between water and plant tissues across a thin cuticle. In many cases subsidiary cells are present adjacent to the guard cells. These are specialized epidermal cells which are usually different in size and shape from other epidermal cells. They are usually smaller than the epidermal cells and have more dense cytoplasmic contents and a greater frequency of cell organelles. In grasses and some other species, subsidiary cells are intimately involved in stomatal mechanism. Diffusion of gases through stomata is governed by the two important parameters associated with the stomata, viz., the stomatal frequency and the extent of opening of the stomata. Stomatal frequency is defined as the number of stomata per unit leaf area (no.mm -2 ). Apart from the stomatal frequency, yet another parameter that reflects the stomatal distribution is the stomatal Index. It is the ratio of the number of stomata per unit leaf area to the total number of epidermal cells and is generally expressed in percentage.

Transpiration is the loss of water through areal parts of the plants particularly leaves in the form of water vapour and 95% of the transpiration is through stomata and the rest from cuticle and the lensi cells. Transpiration can be measured in several ways. The most commonly used technique as the whole plant level is the gravimetric technique where in the amount of water loss through transpiration is measured of monitoring the amount of water added by measuring the loss in weight. Another method is the direct measurement using equipments specially designed for this purpose, which steadily include porometers. Transpiration is also being measured using the portable photosynthesis systems. In the laboratory transpiration rate can be measured using a very simple technique as follows,

During photochemical reactions of photosynthesis, takings place in the chloroplast lamellas system, electrons flow from water to NADP resulting in generation of a powerful reductant NADPH. Simultaneously, water is oxidised and O2 oxygen is released. This reaction is known as Hills reaction. The rate of production of NADPH and ATP in the photochemical reaction is directly associated with the oxidation of water the sole electron donor. So the photochemical efficiency can be measured by measuring the rate of oxygen evolution. Since oxygen evolution is a good reflection of the photochemical activity of chloroplast, determination of the rate of oxygen evolution can be conveniently adopted to estimate the photochemical activities. Normally oxygen evolution as a measure of photochemical reactions, is measured either in isolated chloroplasts or in the excised leaf discs using an oxygen electrode. An oxygen electrode is a special form of chemical electrode cell in which the current generated is proportional to the activity of oxygen present. This equipment is widely used for determining the amount of oxygen liberated during the photochemical reaction either in isolated chloroplasts or in leaf bits. Though oxygen electrode is the most frequently used technique to determine the rate of oxygen evolution, there are a few indirect methods to quantify the amount of oxygen evolved. In photosynthesising tissues or in isolated chloroplasts, the rate of oxygen liberation can be determined by using appropriate artificial electron acceptors and donors (artificial electron donor and acceptors are compounds which can substitute the physiological electron transport carriers and helps in understanding the electron transport mechanisms and ATP synthesis). Oxygen is a powerful oxidizing agent which means it readily oxidizes another compound and itself gets reduced to water. This property can be effectively used in qualitative and quantitative determination of oxygen evolution in green leaves exposed to light. The oxygen that is liberated during the photochemical reactions can be determined by using an appropriate organic substance which can be readily oxidized by oxygen.

Photosynthesis is an important physiological process associated with plant growth and productivity. During the process of photosynthesis, the solar energy is trapped and converted into chemical potential which is later used for the reduction of CO2 into carbohydrates. The powerful reductant, NADPH that is needed for carbon reduction is generated by the light driven reactions of the chloroplast lamellar system. Photosynthesis can be summarized as follows.

Growth and differentiation are regulated by a group of organic compounds known as growth regulators. These growth regulators regulate growth not only at the organ level but also in the plant as a whole. They can be broadly defined as organic compounds synthesized in one part of a plant transported to another where at very low concentration they cause a physiological response. There are five major groups of growth regulators, namely, the Auxins, Gibberellins, Cytokinins, Ethylene andAbscisic acid. Endogenously synthesized growth regulators are often referred to as plant growth hormones. However, a number of synthetic compounds eliciting similar response as that of the growth hormones have been identified and extensively used. These synthetic hormones effectively substitute the endogenous hormones to understand the basic mechanisms of hormones and also for commercial use.

Much of the success on modern agriculture dependson the availability of high quality seeds with good genetic potential and proven performance in germination, emergence, and vigorous Vegetative growth. Seed: Seed is a fertilized mature ovule containing an embryonic axis (embryo), stored food material (endosperm) and a protective covering (seed coat or testa). Seed structures: Living embryo, the most important part of seed, consists of two structures (i) embryonic axis and ( ii ) cotyledon(s). Theembryonic axis is composed of three parts namely (i)radical (embryonic root), the hypocotyls (point of attachment of cotyledons) and (iii) plumule (theshootapex with the first true leaves). Thethree parts of embryonic axis are easy to identify in dicots, but in monocots (especially in the family Gramineae) their identification is difficult. In monocots, there is only one cotyledon, which is reduced and modifiedto form the scutellum. The basalsheath of cotyledon is elongated to take the shape of coleoptile,while in somecases (e.g., maize), the hypocotyls is modified to form mesocotyl. The base of hypocotyls sheathing the radical is termed as coleorhiza. Seed development: Thereare manyvariations in the pattern of seeddevelopment when the entire plant kingdom is considered. However, the general processof This includes seed development is almost similar.

Tissue culture refers to the collection of techniques used to maintain or grow of cells, tissues and organs under controlled environment. The German botanist G. Haberlandt (1854-1945) originated the concept of cell culture in which he described his pioneering experiments with isolated plants cells and developed a concept of totipotency (potential of a cell to reproduce entire plant and all of its characteristics). He is considered as father of tissue culture.

Growth and Development are the most fundamental and conspicuous characteristics of all living organisms. According to dictionary, growth is the advancement towards maturity and development is a gradual increase in size. The plant physiological definition of growth is ‘an irreversible increase in mass, weight or volume of a living organism, organ or cell.

Hydroponics is a subset of hydroculture and is a method of growing plants using mineralnutrient solutions, in water, without soil. Terrestrial plants may be grown withtheir roots in the mineralnutrient solution only or in an inert medium, suchas perlite, gravel,mineral wool, expanded claypebbles or coconut husk The techniques on hydroponics are widely used in plant sciences to elucidate several problemson nutrition. Primarily, this has been not only used to evaluate deficiency symptoms of an essential element but also used to assess waters stress effects in crop plants. There has been a considerable controversy whether such responses paralled with field responses. Though these techniques have their limitations, it is certain that it has its relevance in identifying certain genotypes relatively tolerant to some stresses such as water, O2 and nutrition. Without entering to such debatable issue, it can be said thatwherenecessity demands, such techniques would proveuseful in demonstration purposes and evaluation of macronutrient deficiencies in tomato and maize. Thegrowing of plants in nutrient solution is called as water culture or hydroponics. These experiments in nutrient solutions are carried out to study the mineral nutrient relation of plants. By elements required for plant growth, specific deficiency symptoms can be produced in water culture experiments. Thesuccess of these experiments of producing deficiency symptoms depends upon the use of homogenous biological material and purity of culture solution in respect of different plant nutrients. Hydroponics is a subset of soilless culture. Many types of soilless culture do not use the mineral nutrient solutions required for hydroponics. Plants that are not traditionally grown in a climate would be possible to grow usinga controlled environment systemlike hydroponics. NASA has also looked to utilize hydroponics in the space program. Ray Wheeler, plant physiologist at Kennedy Space Center’s Space Life Science Lab, believes thathydroponics will create advances within space travel. He terms this as a bioregenerative life support system.

Plants are exposed to various stresses such as temperature, water, oxygen, salts, nutrition etc, during its growth under dry land condition. Various simple tests have been developed by plant scientist for evaluation of different cultivars against these stresses. These tests are simple to perform in the laboratory with fewer instruments that are commonly available in most of the laboratories in developing countries. These tests have significance in plant water relations and cultivars reliably screened for various stresses. The crops upon which these tests have been performed here are used by the proponents of the tests. These tests are not advised to be performed on crops other than those used by the authors. It probably needs standardization for other crops. The following test has been explained for the benefit of the readers.

Carbohydrates are widely prevalent in the plant kingdom, comprising the mono, di-, oligo-, and polysaccharides. The common monosaccharides are glucose, fructose, galactose, ribose etc. The disaccharides, i.e., the combination of two monosaccharides include sucrose, lactose and maltose. Starch and cellulose are polysaccharides consisting of many monosaccharide residues. Cellulose is the most abundant organic compound on this planet since it forms part of the cell wall in plants. Aldehydes (–CHO) and ketones (= CO) are active groups in carbohydrates. Carbohydrates contain many hydroxyl groups as well. The number of hydroxyl groups varies with the number of carbon atoms. Monosaccharides contain the free aldehyde or ketone group. Some disaccharides have the free aldehyde group (maltose) and some do not have the free ones (sucrose). The polysaccharides, starch and cellulose, are polymers of monosaccharides linked through the active groups. The chemical properties of saccharides vary depending upon the number of hydroxyl groups and the presence or absence of –CHO/= CO groups. These variations are the basis in the development of colour reactions to identify the saccharides. Some simple tests used to identify the presence/absence of certain saccharides are listed below:

Nitrogen is a nutrient and occurs in many forms including ammonia, organic, nitrate and nitriteeach of which may be tested for in a variety of ways. Under normal circumstances, the nitrite form of nitrogen will not be present in large quantities due to its rapid oxidation orconversion to nitrate. The presence of large concentrationsof ammonia in a stream or lake can create a large oxygen demand. This demand is caused by the conversion of ammonia to nitrate. High concentrations of nitrate in waste water treatment plant effluent can cause algae to grow in large quantities. Current status For many years, the protein contentof foods has been determined on the basis of total nitrogen content, while the Kjeldahl (or similar) method has beenalmost universally applied to determine nitrogen content (AOAC, 2000).Nitrogen content is then multiplied by a factor to arrive at protein content. This approach is based on two assumptions: thatdietary carbohydratesand fats do not contain nitrogen, and thatnearly allof the nitrogen in the dietis present as amino acids in proteins. On the basis of early determinations, the average nitrogen (N) content of proteins was found to be about 16 per cent, which led to use of the calculation Nx6.25(1/0.16 = 6.25) to convertnitrogen contentinto protein content.

In chemistry, and especially in biochemistry, a fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides or phospholipids. When they are not attached to other molecules, they are known as “free” fatty acids. Fatty acids are important sources of fuel because, when metabolized, they yield large quantities of ATP. Many cell types can use either glucose or fatty acids for this purpose.

Equipment Adjustable pipette. 20-ml glass beaker. Atomic Absorption Spectrophotometer.

Phytochemicals are chemical compounds that occur naturally in plants (phyto means “plant” in Greek). Some are responsible for color and other organoleptic properties, such as the deep purple of blueberries and the smell of garlic. The term is generally used to refer to those chemicals that may have biological significance, for example antioxidants, but are not established as essential nutrients. Scientists estimate that there may be as many as 10,000 different phytochemicals having the potential to affect diseases such as cancer, stroke or metabolic syndrome. The potential of the phytochemicals have large scale pharmacological and biological activities such as antioxidant constituents (hydrolysable tannins, phenolic acid and flavonoids) of the plant materials for the care of health and protection from coronary heart diseases, cancer, anti-carcinogenic and anti-mutagenic effects. Varieties of herbaceous vegetables are protective against various diseases, particularly cardiovascular diseases. These herbaceous plants and species are harmless sources for obtaining natural antioxidants. Antioxidant constituents can delay or inhibit the oxidation of lipids and other compounds by inhibiting the propagation of oxidation chain reaction (Hussain et al., 2011) Primarily, antioxidant effect is due to phenolic compounds such as phenolic acid, flavonoids and phenolic diterpenes and their mode of action for antioxidant compounds is due to its redox reaction properties which can absorb and neutralize free radicals by quenching singlet and triplet oxygen. Vitamins are organic substances necessary for metabolism. Human diet does not always contain the required amount of vitamins for the normal growth and maintenance of the body function and as such cannot produce enough quantity for their body metabolism, so it can be obtained from fruits, vegetables and foods. Deficiency of vitamins can cause serious human health diseases and sometimes, very small concentrations are required for maintenance of good human health.

The effects of global warming and/or climate change are of concern both for the environment and human life. Evidence of observed climate change includes the instrumental temperature record, rising sea levels, and decreased snow cover in the Northern Hemisphere. According to the IntergovernmentalPanel for Climate Change (IPCC) Fourth Assessment Report, “most of the observed increase in global average temperatures since the mid-20th century is very likely due to the observed increase in [human greenhousegas] concentrations. It is predicted that futureclimate changes will include further globalwarming (i.e., an upward trendin global mean temperature), sea level rise, and a probable increase in the frequency of some extreme weather events. Ecosystems are seen as being particularly vulnerable to climate change. Human systems are seen as being variable in their capacity toadapt to future climate change. To reduce the risk of large changes in future climate, many countries have implemented policies designed to reducetheir emissions of greenhouse gas.

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