Agronomy and crop physiology are pivotal branches of agricultural science that focus on understanding the growth, development, and productivity of crops under varying environmental and management conditions. The integration of theoretical knowledge with practical application is essential to developing a holistic understanding of these disciplines. This lab manual, Hands-on Experiment in Agronomy and Crop Physiology is designed to bridge the gap between classroom instruction and real-world agricultural practices. The primary objective of this manual is to provide students, researchers, and agricultural practitioners with step-by-step guidance to perform key experiments in agronomy and crop physiology. The experiments outlined in this manual have been carefully selected to cover fundamental concepts, modern methodologies, and applications essential for the study of crop science. Each experiment is structured to foster a clear understanding of the scientific principles involved, while emphasizing hands-on learning and analytical skills. The manual is organized into chapters that encompass a wide range of topics, including soil-crop relationships, plant growth analysis, water and nutrient management, and physiological processes such as photosynthesis, transpiration, and nutrient uptake. Special attention has been given to aligning the content with current academic curricula and emerging trends in agricultural research.

In the realm of natural elements, there exist approximately one hundred and three distinct elements. Among them, nearly ninety elements are absorbed by plants. To differentiate essential elements from those that plants can assimilate but are not vital, Arnon (1954) established the following criteria: 1. The plant must be incapable of normal growth or completing its life cycle in the absence of the element 2. The element is specific and irreplaceable by another 3. The element directly participates in plant metabolism By applying these criteria, molybdenum and chlorine are not deemed essential, even though they serve a functional role in plant metabolism. This is because they can be substituted by vanadium and halides, respectively. Subsequently, D. J. Nicholas refined the definition of essential elements, replacing the term “functional or metabolic nutrient” to encompass any mineral element that contributes to plant metabolism, regardless of whether its action is specific.

Introduction The physiological disorder of a mineral nutrient is entirely contingent upon its function within the plant body. Inadequate or excessive supply of any essential element can result in metabolic disruptions, influencing the activities of enzymes, rates of metabolic reactions, and concentrations of metabolites. Alongside alterations in metabolic patterns, severe deficiencies of specific essential elements also give rise to distinctive effects in leaves, stems, roots, blossoms, and fruits. A. Nitrogen Functions: Nitrogen is essential for plants, playing crucial roles in protein synthesis, nucleic acid formation, and chlorophyll synthesis. It is a key component of enzymes, contributing to various metabolic processes. Nitrogen also supports root development, cell structure, and the synthesis of secondary metabolites. Striking a balance in nitrogen levels is vital for maintaining plant health and ensuring optimal growth and productivity. Effective nitrogen management is essential for sustainable agriculture and overall ecosystem well-being.

The growth and productivity of crops depend on various factors, with the nutrient content of plant parts like leaves and stems playing a crucial role. These parts are often used as indicators to assess the nutrient status of plants. Each crop requires essential elements at specific concentrations during different growth stages, known as ‘critical levels’. When the nutrient content falls below these critical levels, plants may display symptoms of deficiency. Nutrient requirements and availability can be evaluated through: i) plant diagnosis, ii) soil analysis, and iii) plant analysis using qualitative or quantitative methods. By conducting these tests, necessary nutrients can be identified and applied to crops to sustain growth and correct deficiencies. While rapid tissue tests offer quick solutions for nutritional problems, quantitative estimation of both plant and soil nutrient concentrations is more economical and provides a long-term strategy for managing nutritional issues. Implementing quantitative estimation allows for informed application of fertilizers, whether as basal or foliar, thus addressing deficiencies effectively and ensuring sustainable crop growth.

The seed yield, along with the rate of application and uptake of nutrients, is used to determine the following parameters for nitrogen or other nutrients: Partial Factor Productivity (PFP) (kg seed/kg N applied): Partial Factor Productivity (PFP) is a measure used in agriculture to assess the efficiency of nutrient use, particularly nitrogen or other essential nutrients, in crop production. It is calculated as the ratio of the seed yield to the rate of nutrient application. The formula for PFP is: *A higher PFP value indicates greater efficiency, meaning that more crop yield is produced per unit of nutrient applied. This is desirable as it implies that the nutrient is being used more effectively by the crop. Agronomic Efficiency (AE) (kg seed/kg N applied): Agronomic Efficiency is a measure used to evaluate the effectiveness of nutrient application in increasing crop yield. It specifically assesses the yield gain per unit of nutrient applied. The formula for AE is:

Assessment of a soil’s fertility status involves an estimation of its available nutrient status i.e., the portion or amount of nutrient directly available in soil for subsequent uptake by crop plant. This exercise commonly referred to as soil testing and is used to arrive at optimum fertilizer application ratio. The need for estimation of available nutrient arises because only a small fraction of what the soil contains is the total nutrient content of the soil. Soil test is calibrated by correlating them with crop response and the result from the basis for making fertilizer recommendations. Estimation of nutrient contents and forms in materials that are involved in nutrient supply and dynamics is a conical step towards planning scientific nutrient management. In this content, both soil and plant testing information come out of the interpretation of analysis assumes a greater value when their concentrations and amounts can relate to soil fertility, nutrient availability, plant growth, yield and quality of the crop produce.

The Dumas combustion method, named after the renowned French chemist Jean-Baptiste Dumas, is a widely recognized analytical technique employed in the determination of the elemental composition of organic compounds. This method involves the complete combustion of a sample in an oxygen rich environment, followed by the separation and subsequent analysis of the resulting combustion products. Principle The Dumas combustion method involves the complete combustion of a sample in the presence of excess oxygen. The nitrogen present in the organic compound is converted into nitrogen gas (N2 ), which is then measured quantitatively. The process can be summarized as follows: i. Combustion: The sample is combusted at high temperatures (typically around 900-1000°C) in the presence of oxygen. This results in the conversion of all organic material into carbon dioxide (CO2 ), water (H2 O), and nitrogen gas (N2 ). ii. Removal of Interfering Gases: The combustion products are passed through various reagents to remove interfering gases such as CO2 and H2 O. iii. Detection of Nitrogen: The nitrogen gas is then measured, often using a thermal conductivity detector (TCD) or another suitable detection method.

Nitrogen, a key component of organic materials like proteins, is analyzed using the Kjeldahl method, which is the global standard for determining protein content in various substances, including human and animal foods, fertilizers, wastewater, and fossil fuels. The Kjeldahl method involves three main steps: A. Digestion B. Distillation C. Titration Nitrogen, making up 1-4% of a plant’s dry weight, is vital for synthesizing chlorophyll, proteins, and other key compounds. Plants with high nitrogen levels turn dark green due to increased chlorophyll. Understanding nitrogen content in plant tissues is crucial for diagnosing deficiencies or toxicity and for effective nutrient management to improve crop production. Determining the nitrogen content in plants is crucial for understanding plant nutrition, assessing the efficiency of fertilizer use, and making informed decisions in agricultural practices. Various methods can be used to determine nitrogen in plant tissues, with the most common ones being the Kjeldahl method, the Dumas method, and near-infrared reflectance spectroscopy (NIRS).

Sample Digestion Dry ashing and wet oxidation are two widely adopted methods for releasing mineral elements from plant tissues. Dry ashing, typically conducted at temperatures between 550 and 600 °C, can lead to the loss of phosphorus (P) and potassium (K) through volatilization. This method is more time consuming and has several drawbacks, such as the volatilization of sulfur (S) and chlorine (Cl), which can be mitigated by adding sodium carbonate (Na2 CO3 ). Additionally, some phosphorus and micronutrients may become occluded during the process, making dry ashing less commonly used. In contrast, wet oxidation employs oxidizing acids, such as a mixture of nitric acid and perchloric acid (HNO3-HClO4 ) or a tri-acid mixture of nitric acid, sulfuric acid, and perchloric acid (HNO3-H2 SO4-HClO4 ). The use of perchloric acid (HClO4 ) prevents the volatilization loss of potassium (K) and provides a clear solution, while sulfuric acid (H2 SO4 ) aids in completing the oxidation. Upon heating, perchloric acid dissociates into nascent chlorine and oxygen, enhancing the oxidation efficiency at high temperatures. However, direct contact between perchloric acid and plant samples can lead to explosions and f ires; thus, pre-digestion of samples is preferred.

Sample Digestion Dry ashing and wet oxidation are the two widely adopted methods for the release of mineral elements from plant tissues. Dry ashing is carried out usually at an ignition of 550 to 600 °C leads to loss of P and K by volatilization. Dry ashing being comparatively more time taking and also has a few drawbacks (such as S and Cl is lost by volatilization during ignition which can be prevented by adding Na2 CO3 ; a part of P and micronutrients also get occluded) is hence occasionally adopted. On the other hand, wet oxidation employs oxidizing acids like HNO3-HClO4 di-acid or HNO3-H2 SO4-HClO4 tri-acid mixture. The volatilization loss of K is prevented by the use of HClO4 and provides a clear solution while H2 SO4 helps completing oxidation. On heating HClO4 dissociates into nascent chlorine and oxygen, increasing the oxidation efficiency at high temperature. Explosion and fire may occur if there is direct contact of HClO4 with plant sample; hence the pre-digestion of samples is preferred. Digestion with HNO3-HClO4 instead of tri-acid mixture is also adopted specially when S is also to be determined in the same digest. The wet oxidation method being less time-consuming, easier and convenient is given below:

Objective To determine the electrical conductivity (EC) of soil, which is an indicator of the soil’s salinity and its ability to conduct electricity. This provides valuable information about the nutrient availability and potential toxicity to plants. Materials Required • Soil sample • Distilled water • Electrical conductivity meter (EC meter) • Beakers (100 mL) • Measuring cylinder (50 mL) • Stirring rod • Filter paper and funnel • Mortar and pestle (for soil grinding, if necessary)

Objective To determine the pH of a soil sample, which provides insight into the soil’s acidity or alkalinity, influencing nutrient availability, microbial activity, and plant growth. Materials Required • Soil sample • Distilled water • pH meter or pH paper • Beakers (100 mL) • Measuring cylinder (50 mL) • Stirring rod • Filter paper and funnel (if needed) • Mortar and pestle (for soil grinding, if necessary)

The Walkley-Black method is a widely used chemical procedure to estimate soil organic carbon (SOC). It involves the oxidation of organic carbon by potassium dichromate in the presence of sulfuric acid, followed by titration to determine the amount of unreacted dichromate. Here is a detailed step-by-step guide: Materials Required • Soil sample (air-dried and sieved through a 2 mm sieve) • Potassium dichromate solution (1 N) • Concentrated sulfuric acid (H2 SO4 ) • Ferrous ammonium sulfate solution (0.5 N) • Orthophosphoric acid (H3 PO4 ) (optional) • Diphenylamine indicator or N-phenylanthranilic acid (indicator) • Distilled water • Burette, pipette, and conical flasks • Heating mantle or hot plate

Chlorophyll content serves as a vital indicator of plant health and photosynthetic efficiency, with higher levels reflecting robust photosynthesis and overall plant vigor, while lower levels may signal nutrient deficiencies, diseases, or environmental stresses. Assessing chlorophyll content helps evaluate plant productivity, as chlorophyll is essential for absorbing light and converting solar energy into chemical energy. Additionally, the chlorophyll-a to chlorophyll-b ratio provides insights into how plants adapt to various light conditions, revealing their light absorption efficiency and ability to cope with environmental changes. Measuring chlorophyll on a fresh weight, dry weight, and leaf area basis allows researchers and agronomists to gauge the effectiveness of agricultural practices such as fertilization, irrigation, and crop management strategies. It also aids in monitoring the impact of stress factors like drought, salinity, pollution, or pest attacks, which is crucial for developing strategies to enhance crop resilience. Reagents: 80% acetone: - 80 ml acetone + 20 ml water = 100 ml Procedure for Chlorophyll Extraction from Leaves 1. Sample Collection: Collect fresh, healthy leaves from the plant to be analyzed. Ensure that the leaves are free from dust, pests, and diseases. 2. Preparation of Leaf Samples: Rinse the leaves thoroughly with distilled water to remove any surface contaminants. Pat them dry with a clean paper towel. Weigh an exact amount (usually around 0.1-0.5 grams) of fresh leaf tissue using an analytical balance. Record the fresh weight accurately.

Introduction Carotenoids are pigments found in plants that contribute to photosynthesis and provide various health benefits. Measuring carotenoid content in leaves can help assess plant health, productivity, and the effects of environmental conditions. The determination of carotenoids typically involves extraction followed by spectrophotometric analysis. Materials and Equipments • Fresh leaf samples • Acetone, hexane, or a mixture of acetone and hexane (for extraction) • Mortar and pestle or homogenizer • Centrifuge and centrifuge tubes • Spectrophotometer • Volumetric flasks and pipettes • Analytical balance

Leaf Relative Water Content (RWC) is a key indicator of plant water status and is used to assess water stress, physiological health, and the efficiency of water use in plants. It reflects the amount of water in the leaf relative to its maximum water capacity and is a useful measure in plant physiology and stress studies. Materials and Equipment • Fresh leaf samples • Analytical balance • Oven or drying equipment • Desiccator • Caliper or ruler • Distilled water • Petri dishes or similar containers • Weighing boats or paper

Analytical Reagents Chemical reagents are supplied in different grades and each grade has a distinct purpose and range of uses. The purest grade is analytical reagent (AR), the second is laboratory reagent (LR), the third is guaranteed reagent (GR) and fourth is technical grade. Second and third form of reagents have a lot of impurities. Therefore, for the determination of micronutrients, AR grade reagents should be used. Distilled Water Distilled water is always used in chemical analysis. The quality of distilled water varies from single distilled water to double or triple distilled water depending upon the requirement of analytical technique(s) e.g. double distilled water (glass distilled) is always recommended for micro nutrient and heavy metal analysis.

Buffers Bronsted and Lowry defined acids as substances which are able to donate protons, and bases as substances which accept protons. In the above example, BH is an acid because it donates proton and B– is an anion liberated by the deprotonation of the acid. B– behaves like a base, so it is called conjugate base. Acids can be classified into strong acids and weak acids. 1. Strong acids get dissociated almost completely, e.g., hydrochloric acid, sulphuric acid. This is because the conjugate bases of these acids are very weak (have less affinity for the proton). 2. Weak acids get dissociated partially, e.g., acetic acid, carbonic acid. This happens because the conjugate bases of these acids are strong (have greater affinity for proton). Since the dissociation of the weak acids is partial, the equilibrium constant for the dissociation reaction of the weak acid (BH) can be written as follows:

Volumetric Analysis Precautions 1. Use Clean Glassware: Ensure all glassware, such as burettes, pipettes, and volumetric flasks, is thoroughly cleaned and rinsed with distilled water to avoid contamination and ensure accurate measurements. 2. Clean Glassware Properly: Before use, all glass apparatus should be thoroughly cleaned with a cleansing mixture of potassium dichromate (K2 Cr2 O7 ) and concentrated sulfuric acid (H2 SO4 ) to remove any residual contaminants. After cleaning, rinse the glassware thoroughly with distilled water to ensure no traces of the cleansing mixture remain. 3. Proper Calibration: Calibrate all measuring instruments, including burettes, pipettes, and balances, before use to maintain accuracy in measurements. 4. Avoid Parallax Error: When reading the meniscus level in a burette or volumetric flask, ensure the eye is at the same level as the meniscus to prevent parallax error. 5. Use Correct Indicators: Choose the appropriate indicator for the titration based on the type of reaction and the pH range of the endpoint to ensure precise determination of the endpoint.

The concentration of a true solution refers to the amount of solute present in a given quantity of solvent or solution. It is an important property because it determines the solution’s behavior in chemical reactions, its physical proper ties (such as boiling and freezing points), and its utility in various applications. Concentration can be expressed in several ways, depending on the context and the precision required. Methods to Express Concentration of a True Solution 1. Molarity (M) • Definition: Molarity is the number of moles of solute dissolved in one liter (1,000 mL) of solution.