T Y BSc. Botany (Sem V)

TY BOT (Paper V) SBO504 Sem V    CURRENT TRENDS IN PLANT SCIENCES II

Unit I: Ethnobotany and Mushroom Industry

Ethnobotany-

Ø  Definition:

 “Ethnobotany is the study of a region's plants and their practical uses through the traditional knowledge of a local culture and people”.

 Ethnobotany is a multidisciplinary field of study that explores the dynamic relationship between humans and plants. It involves the scientific investigation of how different cultures and societies interact with plant resources for various purposes, including food, medicine, shelter, clothing, ritual, and cultural practices.

Ethnobotanists examine traditional knowledge systems, indigenous practices, and historical records to understand the uses, management, conservation, and cultural significance of plants in different societies. The discipline combines elements of botany, anthropology, ecology, pharmacology, and other related fields to gain insights into the intricate connections between plants and human cultures.

Ø  History:

 The history of ethnobotany dates back thousands of years, with early human societies relying on plants for sustenance, healing, and cultural practices. Here is a brief overview of the history of ethnobotany:

The term "ethnobotany" was coined in 1895 by J.W. Harshberger to encompass the study of plants used by primitive and aboriginal people. Richard Evans Schultes is known as the "father of ethnobotany."

The use of plants by humans for various purposes can be found in ancient Sanskrit, Greek, Arabic literatures, ethnographies, and travelogues.

Ancient Indian Sanskrit literature mentions a variety of plants used in worship, medicine, food, fuel, and agriculture.

Different varieties of Rudraksh (a type of seed) are mentioned in Laxmipuran and Shivpuran.

The Sushruta Samhita and Charak Samhita mention around 1200 plant drugs and their therapeutic applications.

Ancient Times: The use of plants by humans for various purposes can be traced back to ancient civilizations such as the Egyptians, Greeks, Romans, Indians, and Chinese. These societies documented the uses of plants in their traditional texts, including religious scriptures, medicinal treatises, and agricultural manuals.

Indigenous Knowledge: Indigenous cultures around the world possess rich knowledge about the local flora and their uses. They have developed sophisticated systems of plant classification, medicinal plant knowledge, and sustainable harvesting practices over generations.

Early Explorations: With the Age of Exploration in the 15th century, European explorers encountered new plants and traditional plant uses during their voyages. This led to the exchange of knowledge between different cultures and the discovery of valuable medicinal plants, spices, and food crops.

Colonial Era: During the colonial period, European powers colonized various regions and documented the plant resources and traditional knowledge of the indigenous populations. This marked the beginning of systematic ethnobotanical studies, with explorers and botanists recording the uses of plants by local communities.

Emergence as a Discipline: Ethnobotany as a formal discipline began to take shape in the late 19th and early 20th centuries. Scholars such as Richard Spruce, J.W. Harshberger, and Richard Evans Schultes conducted extensive ethnobotanical research in different parts of the world, studying the relationships between plants and indigenous cultures.

Modern Ethnobotany: In the latter half of the 20th century, ethnobotany gained recognition as an interdisciplinary field of study. It drew from various disciplines, including botany, anthropology, pharmacology, ecology, and conservation. Ethnobotanical research expanded to encompass a wide range of topics, such as traditional ecological knowledge, medicinal plant discovery, sustainable resource management, and cultural preservation.

Current Trends: Today, ethnobotany continues to evolve as researchers explore the intricate connections between plants, people, and culture. There is growing emphasis on understanding traditional knowledge systems, fostering community engagement, promoting conservation of plant diversity, and exploring the potential of traditional medicine for modern healthcare.

Sub-disciplines of Ethnobotany:

 Ethnomycology: The study of folk knowledge and uses of mushrooms and other fungi.

Ethnoscience: The study of how different cultures perceive and categorize the world, including folk taxonomy and classification systems.

Ethnomedicine: The study of traditional medicinal practices and remedies, encompassing written systems like Ayurveda and oral traditions of various cultures.

Ethnopharmacology: The study of the uses, effects, and mechanisms of action of naturally-occurring drug compounds, often explaining the effectiveness of herbal medicine.

Ethnomusicology: The study of the music, musical instruments, and dance of different cultures, including their connections to plants.

Ethnoecology: The study of how different cultures perceive and manage the ecosystems they inhabit.

Ethnotoxicology: The study of the use of toxic plants in human societies, such as fish poisons and arrow poisons.

Archaeoethnobotany: The study of plant materials from archaeological sites, combining ethnology, archaeology, and botany to understand human migration, crop origins, and domestication.

Ethnogynaecology: The study of women's health and related practices in tribal societies, including issues of fertility, conception, abortion, and the use of abortifacients.

Ethnonarcotics: The study of the use of narcotics, hallucinogens, and snuffs in primitive societies.

Ethnogastrology: The study of ethnic groups and their food habits, including traditional recipes.

Ethnopaediatrics: The study of healthcare practices specific to children in different ethnic groups.

Ethnoorthopaedics: The study of traditional knowledge and practices related to setting and healing of bones in different ethnic tribes or communities.

Ethnohorticulture: The study of the management and cultivation of useful plants, including fruits, vegetables, and ornamentals in home gardens or orchards.

Ethnoforestry: The study of human management of forests and forest trees.

Ethnoagroforestry: The study of land management practices that involve the simultaneous production of food crops and trees.

Ethnolinguistics: The study of linguistic terminology for plants and plant parts across different language groups.

Ethnosilviculture: The practice of rearing plants of economic and cultural value.

Ethnozoology: The study of the complex relationships between people and animals, including domesticated animals and the management of wild animals for hunting and other uses.

Ethnoentomology: The study of the relationships between people and insects, including their use in rituals, beliefs, pest control, and the utilization of beneficial insects and their products.

Ø  Sources of Data and Methods of Study in Ethnobotany:

I. Fossils:

Plant remains from the past are important tools for collecting and identifying plants used by humans in prehistoric and protohistoric periods.

Paleobotanists conduct specialized studies known as paleo-ethnobotanical studies to analyze and document ancient plant usage.

These studies often include research on the origin and history of agriculture.

Fossilized animal droppings (corpolites) are analyzed to determine the diets of animals in ancient times.

Institutions like the Birbal Sahni Institute of Paleobotany in Lucknow, India, excel in such studies.

II. Archaeological Resources:

Stone inscriptions and sculptures from different time periods may contain information about plants and their uses in ancient times.

Scholars and paleobotanists can identify and interpret plant-related content in archaeological material.

The Archaeological Survey of India is a valuable resource for identifying such material.

III. Folk-Songs, Folk-Tales:

Folk songs, folk tales, and proverbs often contain references to plants, highlighting their characteristics, uses, and cultural associations.

Sociologists and specialists in human history and culture conduct fieldwork to gather this information, while botanists aid in plant identification.

IV. Obsolete Literature:

Neglected documents in libraries, museums, and private collections can provide interesting data on economic uses and relationships of plants.

Critical search for these documents, often referred to as "Grey literature," can uncover valuable ethnobotanical information.

Difficulties in plant identification make the work of an ethnobotanist important and intriguing.

V. Reports of Forest Departments:

Annual reports and working plans prepared by Forest Departments provide information on the commercial value and utilization of various plant species.

Scrutinizing old and new reports can yield additional useful data.

VI. Sanskrit Literature:

Researchers have conducted preliminary studies on floras mentioned in Sanskrit literature, listing over a thousand plant names from texts like the Rigveda, Atharvaveda, Ramayana, Mahabharata, and various shastras.

VII. Ayurvedic Literature:

Ancient Ayurvedic texts like Sushruta Samhita and Charak Samhita mention numerous medicinal plants. These texts provide information on the use and administration of herbal drugs, including their specific applications.

VIII. Ethnographies:

Ethnographers often extensively document the plants used by different tribal groups, providing valuable information for ethnobotanical studies.

Ethnographies written by qualified anthropologists offer scientific insights into the relationships between cultures and plants.

IX. Gazetteers:

District gazetteers highlight important plants in specific regions, offering insights into their local significance.

X. Flora:

Regional floras at the country, state, district, or smaller scale occasionally mention the use of specific plant species, aiding in ethnobotanical evaluations of the area.

XI. Herbaria and Museums:

Herbarium sheets and museum specimens, with their collectors' notes, provide firsthand information on plant locality, people, and time.

Herbaria, such as the Central National Herbarium of the Botanical Survey of India, contain a wealth of ethnobotanical data from collected specimens.

XII. Fieldwork:

Traditional knowledge and ancient practices related to natural resources still exist in various locations.

Personal observation, interaction with reliable informants and local experts, and collection of voucher specimens are essential in studying the knowledge of plants and their usage by local communities.

Applications of ethnobotany:

 Ethnomedicines:

 Ethnomedicine refers to a wide range of healthcare systems, practices, beliefs, and therapeutic techniques that arise from indigenous cultural development.

It involves studying the cultural perspective on disease/illness, indigenous beliefs about signs and symptoms, disease progression, and methods of management.

Ethnomedicinal studies have been conducted in various regions, resulting in interesting therapeutic applications of numerous plant species.

For example, the Bhoxas tribal people of U.P. use ethnomedicine to treat diphtheria and piles.

Native phytotherapy is used for children and women's diseases in Assam.

Ethnomedicines are utilized in postnatal care for women in Assam.

Research in the Gwalior Forest Division, Madhya Pradesh, India, has led to the discovery of therapeutic applications for 102 plant species.

Ethnomedicinal plants are used to treat various diseases, and information on medicinal claims is collected from tribal people and traditional healers.

Studies document the botanical identity, local name, parts used, preparation methods, administration, and diseases treated by specific plants.

Ethnobotanical surveys have been conducted in different regions, such as the Southern Western Ghats of India, to collect information on medicinal plant usage.

Indigenous communities, such as the Paliyars, utilize specific plants for medicinal purposes, listing their Latin names, families, local names, parts used, preparation methods, and medicinal uses.

Ethnomedicinal plants are commonly used to treat skin diseases, poison bites, stomachaches, and nervous disorders.

Tropical rainforests are valuable repositories of medicinal plant species and indigenous ethnomedical knowledge.

Agriculture:

Indigenous agricultural practices are adapted to local conditions and resource availability.

Local techniques of farm management conserve natural resources, rely on local resources, and ensure sustainable production.

Indigenous agricultural systems emphasize the recycling of farm-produced resources and the use of renewable products.

Interdependence between crops, livestock, forests, and fodder is crucial in indigenous farming systems.

Livestock, forests, and crops contribute to the synthesis of Farm Yard Manure (FYM), which serves as a major source of plant nutrients.

Indigenous knowledge plays a significant role in resource-poor agriculture, contributing to sustainable agricultural practices.

Indigenous agriculture provides household-level food security through the optimal use of local resources.

Indigenous agriculture relies on local cultures and the understanding, sharing, and ownership of different resources.

Edible Plants:

Wild edible plants have been identified since prehistoric times and include weeds in urban areas and native plants in wilderness areas.

Edible plants have one or more parts that can be gathered at the appropriate stage of growth and properly prepared for food.

Various plant items, including leaves, tender shoots, and flowers, are consumed as food in tribal regions and sometimes used as medicine.

Edible flowers have specific medicinal properties and are in high demand globally.

Mushrooms have been traditionally used as food, medicine, for mythological purposes, and aesthetics.

Some species of mushrooms are edible and are used in ethnomedicine.

Local people gather wild plants, including fruits, vegetables, and relishes, to meet their daily nutritional needs.

Intensive processing may be required for the consumption of certain wild food plants.

Examples of edible plants include Allium bakeri, Alpinia bracteata, Artocarpus chaplasha, Bauhinia purpurea, Callicarpa arborescens, Castanopsis indica, etc.

In Assam, a total of 35 species of edible flowers belonging to 23 families have been recorded. These flowers not only serve as a source of food but also possess specific medicinal properties, making them highly sought after globally.

Traditional knowledge about edible plants is passed down through generations and is essential for sustainable food sources in indigenous communities.

Indigenous people have identified and utilized at least 15 different edible mushroom species, which are not only consumed as food but also hold cultural and medicinal significance.

Ethnomedicinal uses of mushrooms have been documented, with species belonging to genera such as Termitomyces, Auricularia, Agaricus, Daldinia, Dictyophora, Pleurotus, Russula, Trametes, Chlorophyllum, and Ganoderma.

Some wild food plants require intensive processing before consumption, such as certain species containing calcium oxalate like Arisaema.

Local communities, such as those in the Manang district of Nepal, rely on wild edible plants to meet their daily nutritional needs. These plants are gathered from the surrounding natural environment and provide a valuable source of food.

Edible wild plant use is deeply rooted in local knowledge, and information about these plants is often obtained through interviews with knowledgeable villagers.

In total, 41 plant species have been identified as sources of fruits, juices, vegetables, and local relishes (achar).

The utilization of edible plants highlights the diverse range of resources available in nature and the importance of traditional knowledge in sustainable food systems.

Traditional medicines used by tribal’s in Maharashtra towards

Mushroom industry:

Detail general account of production of mushrooms with respect to methods of Composting, spawning, casing, harvesting of mushroom.

Mushroom farming is one of the most profitable agri-business that you can start with a low investment and less space. Mushroom farming in India is growing gradually as an alternative source of income for many people. Worldwide, US, China, Italy and Netherlands are the top producers of mushrooms. In India, Uttar Pradesh is the leading producer of mushrooms followed by Tripura and Kerala.

Types of Mushrooms

There is various type of edible mushroom available in the world but in India mostly four type mushroom cultivated.  

1.      White Button Mushroom

2.      Portobello Mushroom

3.      Dhingri (Oyster) Mushroom

4.  Paddy Straw Mushroom

Among all above White Button mushroom has high demand the most popular hence most farmer select this variety for commercially mushroom farming.

Average price for white button mushroom is in between 50-100 rs per kg this depends upon market demand. White Button mushroom is mostly consumed mostly hotels and metro cities.

Cultivation Procedure has five main steps.

1.      Mushroom Spawn

2.      preparing of compost

3.      Spawning of mulch

4.      Casing

5.  Cropping and harvest administration

Mushroom Spawn:

Spawn is planting material for mushroom cultivation that is it is a seed of mushroom. For the preparation of mushroom spawn required greater technical skill & investment mostly mushroom spawn produce large institute.

Good Qualities mushroom spawn has following qualities

1.      The spawn  should Be rapidly growing in the compost

2.      Provide early pruning following casing

3.       high yielding

4.  It must create the greater grade of mushroom

 Preparing of compost

 Compost is an artificially prepared growth medium from which mushroom can derive essential nutrients necessary for growth.

There are two primary methods for compost preparation:

·         Longer Method

·         Short Method

Short Method takes less time to prepare compost than longer method but requires more capital and resources.  The compost made by the short method is suitable for high yielding mushroom production.

Short Method  

Compost prepared by this method gives the high-quality product, and there is very little chance of infections.

Wheat straw           300 kg

Wheat bran            15 kg

calcium ammonium nitrate      9 kg

Urea            4 kg

Muriate of Potash      3 kg

Superphosphate       3 kg

Gypsum    20 Kg

Longer Method:

This is an outdoor procedure and takes around 28 days in its conclusion with a total of seven turnings.

Materials are required for the longer method is as follows.

Wheat straw  1000 kg

Chicken manure   600 kg

Wheat bran   60 kg

Urea   15 kg

Gypsum  50 Kg

 Spawning

The seeds are a mix of the compost.  Before seeding, wash the utensils used in seeding and seeding in 2% formalin solution and wash the hands of the person working in the seedling with soap so that any infection can be avoided.

After this, add seed to 0.5 to 0.75 percent, that is, 100 kg G 500-750 grams of seeds are sufficient for ready compost.

Casing soil

The importance of casing soil would be to keep the moisture content and exchange of pollutants inside the top layer of the compost which assists in the correct development of the mycelium. The pH of this casing soil should be 7.5-7.8 and have to be free of any disease.

The casing soil is stacked on the cemented ground and can be treated with 4% formalin solution. Through rotation of the ground is completed and it’s covered with polythene sheet for another 3-4 days. Pasteurization of shell soil at 65° C for 6-8 hours is shown to be a lot more successful.

3-4cm thick coating of casing soil has been spread thickly on the compost once the surface was coated with white mycelium of this fungus. Formalin solution (0.5%) is subsequently being sprayed. Appropriate ventilation ought to be organized together with water being sprayed a couple of times every day.

Harvesting of crop

 Mushroom Pinhead initiation starts after 10-12 days and mushroom crop harvested in 50-60 days.

Harvest Mushrooms by light twisting without bothering the casing soil and  When the harvesting is finished then fill the gap on beds with fresh, sterilized casing material and spray water.

The crop should be harvested before the gills available because this might diminish its quality and market worth.

Post-harvest management

Harvested mushrooms softly wash with  5g. KMS  solution in ten litter water. After washing remove excess water and pack these mushroom in the polythene bag. the package practices depend upon market & you customer demand

Pest & Diseases

The insect pests mostly observed are nematodes, mites and springtails. The crop is suspect to several diseases like Dry Bubble (brown spot), Wet Bubble (White Mould), Cobweb, Green Mould, False truffle (Truffle disease), Olive green mould, Brown plaster mould and Bacterial blotch. Professional help and extension advice will have to sought by the entrepreneur to adopt appropriate and timely control measures against pests & diseases.

Cultivation of Pleurotus, Agaricus, Volvariella mushroom.

Cultivation of Pleurotus (Oyster Mushroom), Agaricus (Button Mushroom), and Volvariella (Straw Mushroom) involves specific cultivation techniques. Here's a general account of the cultivation process for each mushroom species:

Pleurotus (Oyster Mushroom) Cultivation:

Substrate Preparation: Pleurotus mushrooms can be cultivated on various substrates, including agricultural waste materials like straw, sawdust, or a combination of both. The substrate is typically chopped, soaked, and then pasteurized or sterilized to eliminate competing organisms.

Spawn Inoculation: After substrate preparation, it is mixed with spawn (vegetative mycelium) of Pleurotus mushrooms. The spawn colonizes the substrate over a period of time, forming a network of mycelium.

Incubation: The inoculated substrate bags or containers are placed in a controlled environment with appropriate temperature and humidity to allow the mycelium to grow and colonize the substrate.

Fruiting: Once the mycelium has fully colonized the substrate, the bags or containers are exposed to specific environmental conditions, including temperature, humidity, and light. These conditions trigger the formation of fruiting bodies (mushrooms), which grow from the substrate. Proper air circulation and moisture levels are maintained during the fruiting stage.

Harvesting: The mushrooms are harvested when they reach the desired size and maturity. The harvesting process involves carefully cutting or twisting the mushrooms at the base of the stem, taking care not to damage the mycelium or other developing mushrooms.

Agaricus (Button Mushroom) Cultivation:

Composting: Agaricus mushrooms are typically cultivated on composted substrates, which consist of a mixture of organic materials such as straw, horse manure, gypsum, and other supplements. The composting process involves creating optimal conditions for decomposition and microbial activity to transform the raw materials into a nutrient-rich substrate suitable for mushroom growth.

Spawning: Once the composting process is complete, the compost is mixed with mushroom spawn. The spawn is evenly distributed throughout the compost, either manually or using specialized machinery.

Casing: After spawning, a layer of casing material (often a mixture of peat moss, vermiculite, and limestone) is applied on top of the compost. The casing layer provides moisture retention and helps induce the formation of mushroom primordia.

Fruiting and Harvesting: The growing environment is carefully managed, with controlled temperature, humidity, and air circulation. Mushroom primordia develop and grow into mature mushrooms. Harvesting is done by gently twisting or cutting the mature mushrooms at the base of the stem.

Volvariella (Straw Mushroom) Cultivation:

Substrate Preparation: Volvariella mushrooms are typically cultivated on rice straw. The straw is chopped into short lengths and immersed in water for several hours to hydrate and soften it.

Spawning: The hydrated straw is mixed with Volvariella mushroom spawn, ensuring even distribution of spawn throughout the substrate.

Incubation: The inoculated straw is packed into containers or bags and kept in a warm and humid environment for mycelium colonization. Incubation may take several weeks, during which the mycelium grows and spreads through the straw substrate.

Fruiting and Harvesting: Once the mycelium has fully colonized the substrate, the containers or bags are transferred to a cooler environment with reduced humidity and increased light. This change in environmental conditions triggers the formation of mushroom clusters. The mushrooms are harvested by cutting or twisting them at the base when they reach the desired size.

General account of mushrooms: Nutritional value, picking and packaging, economic importance.

Nutritional Value of Mushrooms

Mushrooms contain more protein than fruits  & vegetable and, Mushrooms can also be low in cholesterol.

Apart from their protein content, mushrooms can also be high in certain vitamins like B, C, vitamin D, riboflavin, thiamine nicotinic acid.

Also an excellent source of iron, Potassium, and potassium along with folic acid, a component known for improving the blood and avoidance deficiencies.

Low in Calories and Fat: Mushrooms are naturally low in calories and fat, making them a healthy food choice. They provide a satisfying and flavorful addition to meals without significantly adding to calorie or fat intake.

Carbohydrates: Mushrooms contain carbohydrates, primarily in the form of dietary fiber. The carbohydrate content in mushrooms is relatively low compared to other plant-based foods, making them suitable for low-carb diets.

Protein: Mushrooms are a good source of plant-based protein. While the protein content may vary among different mushroom species, they generally provide a decent amount of protein compared to most vegetables.

Vitamins: Mushrooms are rich in various vitamins, especially B vitamins. They are an excellent source of riboflavin (vitamin B2) and provide good amounts of other B vitamins such as thiamine (B1), niacin (B3), and pantothenic acid (B5). B vitamins are essential for energy production, metabolism, and overall cellular function.

Minerals: Mushrooms contain several essential minerals. They are a source of potassium, which is important for maintaining healthy blood pressure and heart function. Mushrooms also provide minerals like phosphorus, which is crucial for bone health, and zinc, which plays a role in immune function and wound healing.

Antioxidants: Mushrooms are known to be rich in various antioxidants, including selenium and ergothioneine. Antioxidants help protect the body against oxidative stress and may have potential health benefits, including reducing the risk of chronic diseases.

Vitamin D: Some mushrooms, particularly those exposed to ultraviolet (UV) light, can produce vitamin D. Vitamin D is important for bone health, immune function, and overall well-being. However, the amount of vitamin D produced in mushrooms is generally lower compared to other food sources like fatty fish or fortified dairy products.

Water Content: Mushrooms have a high water content, which contributes to their low calorie density. This can help with hydration and provide a feeling of fullness when consuming them.

The nutritional value per 100 grams of mushrooms is as follows:

Energy: 113 kJ (27 kcal)

Water: 92.45 g

Carbohydrates: 4.1 mg

Fat: 0.1 g

Protein: 2.5 g

Thiamine (Vitamin B1): 0.1 mg (9% of recommended daily intake)

Riboflavin (Vitamin B2): 0.5 mg (42% of recommended daily intake)

Niacin (Vitamin B3): 3.8 mg (25% of recommended daily intake)

Pantothenic Acid (Vitamin B5): 1.5 mg (30% of recommended daily intake)

Vitamin C: 0 mg (0% of recommended daily intake)

Calcium: 18 mg (2% of recommended daily intake)

Phosphorus: 120 mg (17% of recommended daily intake)

Potassium: 448 mg (10% of recommended daily intake)

Sodium: 6 mg (0% of recommended daily intake)

Zinc: 1.1 mg (12% of recommended daily intake)

Vitamin D (D2 + D3): 0.2 µg

Sugar: 1.98 g

Picking and packaging of mushrooms: It involve careful handling and storage to maintain their freshness and quality. Here's a general account of the process:

Picking:

Mushrooms are typically hand-picked to ensure proper selection and minimize damage to the delicate fruiting bodies. Skilled pickers carefully harvest mushrooms at the optimal stage of maturity. This stage may vary depending on the mushroom species and the desired market preferences.

Sorting and Grading:

After picking, the mushrooms go through a sorting and grading process. This involves inspecting each mushroom for quality, size, and appearance. Mushrooms with blemishes, bruises, or signs of decay are removed. They are also sorted based on their size and uniformity. This step helps ensure that only high-quality mushrooms move forward in the packaging process.

Cleaning:

Mushrooms are cleaned to remove any dirt, debris, or residual substrate that may be attached to them. This is typically done by gently brushing or wiping the mushrooms with a soft cloth or brush. Some commercial operations may use automated mushroom cleaning systems to streamline the process.

Packaging:

Mushrooms are packaged to protect them during transportation and storage, as well as to provide information to consumers. Packaging options can vary, but common materials include punnets (plastic or cardboard trays), plastic bags, or clamshell containers. These packages are designed to allow air circulation while protecting the mushrooms from physical damage.

Labeling and Storage:

Packages are labeled with relevant information such as the mushroom variety, weight, nutritional information, and producer's details. Proper labeling helps consumers make informed choices. After packaging, the mushrooms are stored in appropriate conditions to maintain freshness. This typically involves storing them in a cool environment with controlled temperature and humidity levels. Some mushroom varieties, such as oyster mushrooms, are more perishable and require specific storage conditions to prolong their shelf life.

Economic importance of Mushrooms:

Mushrooms hold significant economic importance in various sectors and industries. Here's a general account of their economic significance:

Food Industry: Mushrooms are widely consumed and valued as a nutritious and flavorful food. They are used in a variety of culinary applications, including soups, sauces, stir-fries, salads, and as a meat substitute in vegetarian and vegan dishes. The demand for mushrooms in the food industry has been steadily growing, leading to increased cultivation and commercial production.

Agricultural Sector: Mushroom cultivation provides employment opportunities and income generation, particularly in rural areas. It can be an alternative agricultural activity for farmers, allowing them to diversify their income streams. Mushroom farms require relatively small land areas and can be established in both rural and urban settings.

Export and Trade: Many countries engage in the export and trade of mushrooms. Mushroom cultivation offers opportunities for international trade, contributing to economic growth and foreign exchange earnings. Export markets may have high demand for specific mushroom species or varieties, especially gourmet and medicinal mushrooms.

Health and Wellness Industry: Mushrooms are recognized for their potential health benefits and medicinal properties. Certain mushroom species contain bioactive compounds that have been studied for their immune-boosting, antioxidant, anti-inflammatory, and anti-cancer properties. The growing interest in natural and functional foods has led to increased demand for medicinal mushrooms and mushroom-based dietary supplements, contributing to the economic significance of the mushroom industry.

Waste Management and Recycling: Mushroom cultivation can utilize various organic waste materials, such as agricultural residues, spent coffee grounds, sawdust, and straw, as substrates for composting and mushroom production. By converting these waste materials into valuable products, the mushroom industry contributes to waste management and recycling efforts, reducing environmental impact and promoting sustainability.

Job Creation and Entrepreneurship: Mushroom cultivation can create employment opportunities at different levels, including farm labor, management, marketing, research, and product development. It also offers opportunities for entrepreneurship, allowing individuals to start their own mushroom farms or related businesses, such as mushroom spawn production, value-added product manufacturing, or mushroom-based food services.

Research and Innovation: The mushroom industry continues to be an area of research and innovation. Scientists and entrepreneurs explore new cultivation techniques, develop improved varieties, and discover novel uses and applications for mushrooms. This fosters technological advancements, knowledge dissemination, and the development of value-added products, further contributing to the economic importance of mushrooms.

Unit II: Plant Biotechnology I

Construction of genomic DNA libraries:

Principle of Genomic Libraries: A genomic library contains all the sequences present in the genome of an organism (apart from any sequences, such as telomeres that cannot be readily cloned). It is a collection of cloned, restriction-enzyme-digested DNA fragments containing at least one copy of every DNA sequence in a genome. The entire genome of an organism is represented as a set of DNA fragments inserted into a vector molecule.

 Construction of genomic library involves following steps:

 (а) Isolation of target DNA:

1. The first step is the isolation of genomic DNA. The procedures vary widely according to the organism under study.

2. If the intention is to prepare a nuclear genomic library, then the DNA in the nucleus is isolated, ignoring whatever DNA is present in the mitochondria or chloroplasts.

3. Genomic libraries can be constructed by isolation of complete DNA from bacteria, virus, plants and animals.

4. In eukaryotes, high molecular weight DNA is isolated by CTAB or SDS methods. The isolated DNA is then purified by caesium chloride and other methods.

(b) Restriction Fragments:

1. The DNA is then fragmented to a suitable size for ligation into the vector.

2. This could be done by complete digestion with a restriction endonuclease. Digestion by the use of restriction endonuclease produces DNA fragments which are not intact.

3. To solve this problem we use partial digestion with a frequently cutting enzyme (such as Sau3A, with a four-base-pair recognition site) to generate a random collection of fragments with a suitable size distribution.

4. Once prepared, the fragments that will form the inserts are often treated with phosphatase, to remove terminal phosphate groups.

5. This ensures that separate pieces of insert DNA cannot be ligated together before they are ligated into the vector.

(c) Cloning the fragments in vector:

1. In place of phages and plasmids, other vectors are in use for construction of large sized DNA libraries.

2. These include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). These are considered as high capacity vectors. 3. Although they are ideal for construction of gene libraries, there are many practical difficulties associated with their use.

4. This will depend on the kind of vector used. The vector needs to be digested with an enzyme appropriate to the insert material we are trying to clone.

5. All engineered plasmids or expression vectors, have 3 main distinctive features:

➢ an origin of replication

➢ A multiple cloning site (MCS)

➢ A selectable marker (usually antibiotic resistance)

6. In a more stable transformation, the transfered gene can be inserted in the genome of the cell, which guarantees its replication in the cell’s progeny.

7. Usually, a marker gene is co-transformed with the gene of interest, which gives the transformed cell some selectable advantage, such as antibiotic resistance.

List of vectors available for DNA libraries:

·         BAC (Bacterial Artificial Chromosome) (75 - 300 kb) :

·         YAC (Yeast Artificial Chromosome) : (100 - 1000 kb):

·         MAC (Mammalian Artificial Chromosome) (100 - >1000 kb):

·         Plasmid : Multicopy plasmids (<10  Kb)

·         Phage : Bacteriophage Υ : (5-10  kb)

·         Cosmid : Bacteriophage:  Υ cos site (35-35 Kb )

Chromosome libraries:

Chromosome libraries, also known as chromosomal DNA libraries or chromosome-specific libraries, are specialized collections of DNA fragments derived from the chromosomes of a particular organism. These libraries play a critical role in genetic and genomic research, providing a comprehensive representation of an organism's genome. Unlike genomic DNA libraries, which contain random DNA fragments from the entire genome, chromosome libraries are focused on preserving the structural integrity and organization of individual chromosomes.

Construction of Chromosome Libraries:

Isolation of Chromosomes: The first step in constructing a chromosome library involves isolating chromosomes from the target organism. This process can be challenging and requires careful extraction and purification techniques to prevent contamination and maintain chromosome integrity.

Fragmentation of Chromosomal DNA: Once the chromosomes are isolated, the chromosomal DNA is fragmented into manageable sizes. Various methods, such as restriction enzyme digestion or mechanical shearing, can be used to achieve this. It is crucial to control the size range of the DNA fragments to ensure proper representation of each chromosome.

Cloning the DNA Fragments: The fragmented chromosomal DNA is then ligated into suitable vectors, such as bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs). These vectors can accommodate large DNA inserts, making them ideal for capturing the entire genetic information of individual chromosomes.

Transformation and Screening: After ligation, the recombinant vectors containing chromosomal DNA fragments are introduced into host cells, usually bacteria or yeast, through transformation. The transformed cells are then spread on solid media to form colonies, with each colony representing a unique clone containing a specific chromosomal DNA fragment.

To identify clones carrying specific chromosomes of interest, several screening methods can be employed:

a. Fluorescence In Situ Hybridization (FISH): FISH uses fluorescently labeled DNA probes that hybridize to specific chromosomal regions, allowing researchers to visualize and identify clones carrying the targeted chromosome.

b. PCR-Based Screening: Specific primers designed based on known chromosomal sequences can be used to screen the library via PCR, enabling the identification of clones containing the desired chromosome.

c. Southern Blotting: Southern blotting can be used to probe the library with specific DNA sequences, facilitating the identification of clones containing the desired chromosomal fragments.

Applications of Chromosome Libraries:

Genome Sequencing and Assembly: Chromosome libraries are crucial resources in large-scale genome sequencing projects, as they facilitate the sequencing and assembly of entire chromosomes, providing valuable insights into the organization and structure of the genome.

1.      Identification of Disease-Causing Genes: Chromosome libraries are used in disease gene mapping studies to identify genes associated with specific genetic disorders. By screening the library with DNA from affected individuals, researchers can locate the chromosomal regions containing the disease-causing genes.

2.      Comparative Genomics: Comparative studies between different organisms can be performed by constructing chromosome libraries and analyzing the conservation and divergence of chromosomal regions among species.

3.      Gene Function Studies: Chromosome libraries enable the isolation of large DNA fragments containing complete genes, allowing researchers to study gene function, regulation, and expression patterns in their native genomic context.

c- DNA libraries:

Meaning of cDNA Library: A cDNA library is defined as a collection of cDNA fragments, each of which has been cloned into a separate vector molecule.

 Principle of cDNA Library:

➢ In the case of cDNA libraries we produce DNA copies of the RNA sequences (usually the mRNA) of an organism and clone them.

➢ It is called a cDNA library because all the DNA in this library is complementary to mRNA and are produced by the reverse transcription of the m-RNA.

➢ Much of eukaryotic DNA consists of repetitive sequences (non-coding) that are not transcribed into mRNA and the sequences are not represented in a cDNA library. It must be noted that prokaryotes and lower eukaryotes do not contain introns, and preparation of cDNA is generally unnecessary for these organisms. Hence, cDNA libraries are produced only from higher eukaryotes.

 ➢ cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism.

 The construction of cDNA library involves following steps-

1. Extraction of mRNA

2. Synthesis of first and second strand of cDNA

3. Incorporation of cDNA into a vector 4. Cloning of cDNAs

1. Extraction of mRNA from the eukaryotic Cell:

It involves the isolation of total mRNA from a cell type or tissue of interest.

v  m-RNA is separated by using density gradient centrifugation

v  mRNA preparation from specialized cell types, e.g. developing seeds, chicken oviduct, erythrocytes, β cells of pancreas etc. 

v  After centrifugation step  The 3′ ends of eukaryotic mRNA consist of a string of 50 – 250 adenylate residues (poly A Tail) which makes the separation easy in a cell extract using oligo-dTs. 

v  When a cell extract is passed through an oligo-dT, the mRNAs bind to the complementary base-pairing between poly (A) tail and oligo-dT.

v Other RNAs (ribosomal RNAs and transfer RNAs) flow through as unbound fraction. The bound mRNAs can then be eluted using a low-salt buffer.

Synthesis of first and second strand of cDNA

1. mRNA single-stranded It is first converted into DNA before insertion into a suitable vector which can be achieved using reverse transcriptase (RNA-dependent DNA polymerase or RTase) obtained from avian myeloblastosis virus (AMV).

2. A short oligo (dT) primer is annealed to the Poly (A) tail on the mRNA.

3. Reverse transcriptase extends the 3´-end of the primer using mRNA molecule as a template producing a cDNA: mRNA hybrid.

4. The mRNA from the cDNA: mRNA hybrid can be removed by RNase H or Alkaline hydrolysis to give ass-cDNA molecule.

5. No primer is required as the 3´end of this ss-cDNA serves as its own primer generating a short hairpin loop at this end.This free 3´-OH is required for the synthesis of its complementary strand.

6. The single stranded (ss) cDNA is then converted into double stranded (ds) cDNA by either RTase or E. coli DNA polymerase.

7. The ds-cDNA can be trimmed with S1 nuclease to obtain blunt–ended ds-cDNA molecule followed by addition of terminal transferase to tail the cDNA with C's and ligation into a vector.

Incorporation of cDNA into a vector

➢ The blunt-ended cDNA termini are modified in order to ligate into a vector to prepare ds-cDNA for cloning. Since blunt-end ligation is inefficient, short restriction-site linkers are first ligated to both ends.

Linker

➢ It is a double-stranded DNA segment with a recognition site for a particular restriction enzyme. It is 10-12 base pairs long prepared by hybridizing chemically synthesized complementary oligonucleotides. The blunt ended ds-DNAs are ligated with the linkers by the DNA ligase from T4 Bacteriophage

The resulting double-stranded cDNAs with linkers at both ends are treated with a restriction enzyme specific for the linker generating cDNA molecules with sticky ends. Problems arise, when cDNA itself has a site for the restriction enzyme cleaving the linkers.

This can be overcome using an appropriate modification enzyme (methylase) to protect any internal recognition site from digestion which methylates specific bases within the restriction-site sequence, thereby, preventing the restriction enzyme binding.

Ligation of the digested ds-cDNA into a vector is the final step in the construction of a cDNA library.

➢ The vectors (e.g. plasmid or bacteriophage) should be restricted with the same restriction enzyme used for linkers.

➢ The E. coli cells are transformed with the recombinant vector, producing a library of plasmid or λ clones.

➢ These clones contain cDNA corresponding to a particular mRNA.

Identification of specific cloned sequences in c-DNA libraries:

cDNAs are usually cloned in phage insertion vectors. Bacteriophage vectors offer the following advantageous over plasmid vectors

1. More suitable when a large number of recombinants are required for cloning

2. Low-abundant mRNAs as recombinant phages are produced by in vitro packaging.

3. Can easily store and handle large numbers of phage clones, as compared to the bacterial colonies carrying plasmids.

4. Plasmid vectors are used extensively for cDNA cloning, particularly in the isolation of the desired cDNA sequence involving the screening of a relatively small number of clones

Cloning of cDNAs

Commonly used vectors for cDNA cloning and expression:

Lambda gt10, Lambda gt11:  DNA inserts of 7.6 kb and 7.2 kb, respectively, inserted at a unique EcoRI cloning site; recombinant Lambda gt10 selected on the basis of plaque morphology; Lambda gt11 has E. coli LacZ gene: LacZ and cDNA encoded protein is expressed as fusion protein.

Lambda ZAP series (phasmids): Up to 10 kb DNA insert; therefore, most cDNAs can be cloned; polylinker has six cloning site; T3 and T7 RNA polymerase sites flank the polylinker so that riboprobes of both strands can be prepared; these features are contained in plasmid vector p Bluescript, which is inserted into the phage genome; the plasmid containing cDNA recovered simply by co-infecting the bacteria with a helper f1 phage that helps excise from the phage genome

Analysis of genes and gene transcripts:

Analysis of genes and gene transcripts is a crucial aspect of molecular biology and genomics research. Understanding the structure, function, and regulation of genes and their transcripts provides valuable insights into various biological processes, including development, disease, and cellular responses. Here, we will explore some common methods and techniques used in the analysis of genes and gene transcripts.

Gene Identification and Annotation: The first step in gene analysis is the identification and annotation of genes within a genome. Computational methods, such as gene prediction algorithms and sequence homology searches, are employed to locate potential gene coding regions. Experimental validation, including RNA sequencing (RNA-seq) data and cDNA cloning, is often used to confirm gene annotations.

RNA Isolation and Preparation: To study gene transcripts, total RNA is isolated from the cells or tissues of interest. Techniques like TRIzol extraction or column-based RNA purification kits are commonly used for this purpose. The isolated RNA may contain different types of RNA molecules, including mRNA, rRNA, tRNA, and non-coding RNAs.

Reverse Transcription (RT) and cDNA Synthesis: For most gene expression studies, total RNA is reverse-transcribed into complementary DNA (cDNA) using reverse transcriptase enzymes. This step converts RNA into cDNA, which can then be used for further analysis, including quantitative PCR (qPCR), RNA-seq, and cloning.

Quantitative PCR (qPCR): qPCR is a powerful technique used to quantify gene expression levels. It allows researchers to measure the abundance of specific gene transcripts in different samples. This method is commonly used to study changes in gene expression under various conditions, such as during development, in response to stimuli, or in disease states.

RNA Sequencing (RNA-seq): RNA-seq is a high-throughput technique that provides a comprehensive view of the entire transcriptome. It involves sequencing cDNA generated from RNA samples, allowing researchers to identify all expressed genes, alternative splicing events, and non-coding RNAs. RNA-seq is particularly useful for studying gene expression patterns and discovering novel transcripts.

Northern Blotting: Northern blotting is an older but still valuable method for analyzing gene transcripts. It involves the separation of RNA molecules by gel electrophoresis, followed by their transfer to a membrane and hybridization with specific DNA or RNA probes. This technique allows the detection and quantification of specific RNA transcripts.

In Situ Hybridization: In situ hybridization is a powerful tool for studying gene expression patterns in tissues. It involves the use of labeled RNA or DNA probes that hybridize with specific mRNA molecules in fixed tissue sections. This method allows researchers to visualize the spatial distribution of gene transcripts within cells and tissues.

Alternative Splicing Analysis: Alternative splicing is a process that generates multiple mRNA isoforms from a single gene, significantly increasing the diversity of gene products. Analyzing alternative splicing patterns is essential for understanding gene regulation and functional diversity. RNA-seq data analysis and PCR-based methods, such as reverse transcription-PCR (RT-PCR), are commonly used to study alternative splicing events.

Analysis of cloned DNA sequences:

Hybridization:

Hybridization, specifically Southern Hybridization, is a fundamental molecular biology technique used to detect specific DNA sequences within a complex mixture of DNA molecules. This technique is named after its inventor, Edwin Southern, who first described it in 1975. Southern Hybridization is widely used for various applications, such as DNA identification, gene mapping, and the study of DNA rearrangements.

Principle of Southern Hybridization: Southern Hybridization involves the specific binding (hybridization) of a labeled DNA probe to complementary DNA sequences within the target sample. The DNA probe is a short, single-stranded DNA molecule that is complementary to the target DNA sequence of interest. This probe is labeled with a detectable marker, such as a radioactive isotope or a fluorescent tag, which allows the identification of the hybridized DNA molecules.

Steps of Southern Hybridization:

DNA Digestion: The first step in Southern Hybridization is to isolate the DNA from the sample of interest, which can be genomic DNA, plasmid DNA, or DNA fragments from a gel electrophoresis experiment. The DNA is then typically digested with a restriction enzyme, which cuts the DNA at specific recognition sequences, generating smaller DNA fragments.

Gel Electrophoresis: After digestion, the DNA fragments are separated based on their size using gel electrophoresis. The DNA fragments are loaded into wells of an agarose gel and subjected to an electric field. Smaller DNA fragments migrate more quickly through the gel, resulting in distinct bands representing different DNA sizes.

DNA Denaturation and Transfer: Following electrophoresis, the DNA fragments in the gel are denatured to single-stranded DNA by soaking the gel in an alkali solution. The single-stranded DNA is then transferred onto a solid support, such as a nylon or nitrocellulose membrane, using a technique called blotting.

DNA Hybridization: The membrane containing the transferred DNA is now ready for hybridization. The membrane is incubated with the labeled DNA probe, and the probe's single-stranded sequence will hybridize (bind) specifically to complementary sequences present in the DNA fragments on the membrane.

Washing and Detection: After hybridization, the membrane is washed to remove any unbound or nonspecifically bound probe molecules. The bound probe-DNA hybrids are then detected using autoradiography (if the probe is labeled with a radioactive marker) or using a fluorescence scanner (if the probe is labeled with a fluorescent tag). The resulting image shows the presence and positions of the DNA fragments that have hybridized with the probe.

Applications of Southern Hybridization:

v  DNA Identification: Southern Hybridization is used to identify specific DNA sequences in a sample, such as detecting the presence of a particular gene or genetic marker.

v  Gene Mapping: Southern Hybridization is employed in gene mapping studies to determine the organization and location of specific DNA sequences within the genome.

v  DNA Fingerprints: The technique is utilized in DNA fingerprinting to compare DNA samples from different individuals or organisms and determine their genetic relatedness.

v  Analysis of DNA Rearrangements: Southern Hybridization helps in detecting DNA rearrangements, such as deletions, insertions, or translocations, that may occur in genetic diseases or during genetic engineering experiments.

Restriction enzyme:

Restriction enzymes, also known as restriction endonucleases, are a class of enzymes found in bacteria that play a crucial role in molecular biology and genetic engineering. These enzymes have the ability to recognize specific DNA sequences and cleave (cut) the DNA at or near these recognition sites. The discovery of restriction enzymes has revolutionized the field of genetics by enabling the manipulation and analysis of DNA with high precision.

Key Features of Restriction Enzymes:

Specificity: Each restriction enzyme recognizes a specific short DNA sequence known as a recognition site or restriction site. These sequences are typically palindromic, meaning they read the same on both strands when read in the 5' to 3' direction. For example, the recognition site for the restriction enzyme EcoRI is GAATTC, and its reverse complement is CTTAAG.

Cleavage: Once a restriction enzyme recognizes its specific DNA sequence, it cleaves the DNA backbone at specific positions within or near the recognition site. The cleavage can result in either a blunt end or staggered ends known as "sticky ends."

Blunt Ends and Sticky Ends: Some restriction enzymes produce blunt ends, where the DNA is cut straight across both strands, resulting in no overhanging nucleotides. Others produce sticky ends, where the DNA is cut asymmetrically, creating single-stranded overhangs that are complementary to each other. Sticky ends are particularly useful in DNA cloning and recombinant DNA technology.

The three main types of restriction enzymes are Type I, Type II, and Type III. Each type exhibits distinct features and functions in DNA cleavage and modification.

Type I Restriction Enzymes:

Type I restriction enzymes are large, multisubunit enzymes with both restriction and modification activities. They recognize specific DNA sequences but cleave the DNA at a considerable distance (usually over 1000 base pairs) from the recognition site. Moreover, the cleavage site is not specific but occurs randomly within a defined region. Type I enzymes also possess a separate methyltransferase activity that methylates the recognition sequence to protect the host DNA from restriction.

Example: EcoKI is a Type I restriction enzyme that is found in Escherichia coli (E. coli). It recognizes the DNA sequence 5'-AACNNNNN↓NGTGC-3', where "N" denotes any nucleotide and "↓" indicates the cleavage site, which can occur at variable positions within the defined region.

Type II Restriction Enzymes:

Type II restriction enzymes are the most commonly used and well-known group. They are simple, single-subunit enzymes that recognize specific short DNA sequences and cleave the DNA at precise positions within or near the recognition site. Type II enzymes are widely used in molecular biology for DNA manipulation, gene cloning, and genetic engineering.

Example: EcoRI is a Type II restriction enzyme derived from Escherichia coli (E. coli). It recognizes the palindromic DNA sequence 5'-GAATTC-3' and cleaves the DNA between the G and A, generating "sticky ends" with single-stranded overhangs.

Type III Restriction Enzymes:

Type III restriction enzymes are multisubunit enzymes that have both restriction and modification activities. Similar to Type I enzymes, they recognize specific DNA sequences and cleave the DNA at a considerable distance from the recognition site. However, the cleavage occurs within a defined region, like Type II enzymes.

Example: EcoP15I is a Type III restriction enzyme found in Escherichia coli (E. coli). It recognizes the DNA sequence 5'-CAGCAG(N8)CTGCAG-3', and the cleavage occurs 25-27 base pairs downstream from the recognition site.

 Example:

DNA Cloning Using EcoRI

Recognition Site: EcoRI is a commonly used restriction enzyme, derived from the bacterium Escherichia coli (E. coli). It recognizes the palindromic DNA sequence 5'-GAATTC-3' on the double-stranded DNA.

DNA Fragment of Interest: Suppose we have isolated a DNA fragment of interest that contains a gene we want to clone. The DNA fragment has the following sequence:

5'-GATATCGAATTCGATATC-3'

3'-CTATAGCTTAAGCTATAG-5'

EcoRI Digestion: To clone the gene into a plasmid vector, we need to cut the DNA fragment at the EcoRI recognition site. When EcoRI cleaves the DNA, it creates "sticky ends" with single-stranded overhangs:

5'-GATATC GAATTC GATATC-3'

3'-CTATAG CTTAAG CTATAG-5'

Plasmid Vector: We also have a plasmid vector with a multiple cloning site (MCS) containing an EcoRI recognition site:

5'-GATATC GAATTC GATATC-3'

3'-CTATAG CTTAAG CTATAG-5'

DNA Ligation: The DNA fragment and the plasmid vector are mixed together in the presence of DNA ligase, an enzyme that catalyzes the formation of phosphodiester bonds between the DNA fragments. The sticky ends of the DNA fragment and the vector are complementary and can base-pair with each other:

5'-GATATC GAATTC GATATC-3'

3'-CTATAG CTTAAG CTATAG-5'

|

5'-GATATC GAATTC GATATC-3'

3'-CTATAG CTTAAG CTATAG-5'

Applications of Restriction Enzymes:

1.  DNA Cloning: Restriction enzymes are essential tools for DNA cloning. They allow researchers to cut a DNA fragment of interest and ligate it into a vector, such as a plasmid, to create recombinant DNA molecules. These recombinant molecules can then be transferred into host cells, enabling the replication and expression of the cloned DNA.

2.  Gene Editing: In modern gene editing techniques like CRISPR-Cas9, restriction enzymes are used to introduce precise DNA breaks at specific locations in the genome. These breaks trigger the cell's repair mechanisms, which can be harnessed to modify or replace the targeted DNA sequence.

3.  DNA Fragment Analysis: Restriction enzymes are used in DNA fragment analysis to determine the presence or absence of specific DNA sequences. For example, they are employed in Southern blotting and RFLP (Restriction Fragment Length Polymorphism) analysis.

4. DNA Mapping: By digesting genomic DNA with different restriction enzymes and analyzing the resulting fragments, researchers can create a genetic map of an organism's DNA, showing the locations of restriction sites and the distances between them.

5.  Polymerase Chain Reaction (PCR): Some PCR-based techniques, such as Restriction Fragment Length Polymorphism PCR (RFLP-PCR), use restriction enzymes to detect sequence variations in specific genes associated with genetic disorders.

Unit III: Instrumentation

In the world of scientific exploration and industrial analysis, instrumentation serves as the foundation, allowing precise measurement and analysis of diverse properties. Colorimetry and spectrophotometry gauge light absorption to reveal chemical details, while chromatography, including column, adsorption, partition, ion exchange, and molecular sieve chromatography, adeptly separates mixtures. These techniques find applications across fields, from pharmaceuticals to environmental monitoring, offering invaluable insights into matter's composition and behavior.

Colorimetry and Spectrophotometry (Visible, UV and IR):

v  Colorimetry:

Colorimeter is a form of photometer which deals with the measurement of light transmitting power of a colored solution in order to determine the concentration of light absorbing substances present within it. It was invented by Louis J Dubosca in 1870. The concentration of colored solute in a solution is estimated by comparing its color intensity with that of standard solution containing a known concentration of solute.

Principle of Colorimetry: When a beam of incident light of intensity I0 passes through a solution, following events occur:

Ø  A part of incident light is reflected. It is denoted by Ir

Ø  A part of incident light is absorbed. It is denoted by Ia

Ø  Remaining incident light is transmitted. It is denoted by It

As Ir is kept constant by using cells with identical properties, the light that is not absorbed is transmitted through the solution and gives the solution its color. Note that color of the incident light should be complementary to that of color of the solution.

The ratio of the intensity of transmitted light (It) to the intensity of incident light (I0) is called transmittance (T). Photometric instruments measure transmittance. In mathematical terms,                                                               T = It÷I0

The absorbance (A) of the solution (at a given wavelength) is defined as equal to the logarithm (base 10) of 1÷T. That is,

A = log (1÷T)

These measurements are dependent on two important laws:

Beer’s law: When monochromatic light passes through a colored solution, the amount of light absorbed is directly proportional to the concentration (C) of solute in the solution.

Lambert’s law: When monochromatic light passes through a colored solution, the amount of light absorbed is directly proportional to the length (L) or thickness of the solution.

When combining Beer-Lambert’s law,

Absorbance (A) α CL

Or,

A= KCL

Where K is a constant known as absorption coefficient.

As the path length is same (as same cuvette is used), Concentration of an unknown solution can be determined by using equation:

Instrumentation of Colorimetry:

i. Light Source: The light source should produce energy at sufficient intensity throughout the whole visible spectrum (380-780nm). Tungsten lamp is frequently used.

ii. Slit: It allows a beam of light to path and minimize unwanted light.

iii. Condensing lens:Give parallel beam of light.

iv. Monochromator: It is used to produce monochromatic radiation (one wavelength band) from polychromatic radiation (white light) produced from light source. It allows required wavelength to pass through it. Prism, gelatin fibers, grating monochromators or interference filters can be used.

v. Sample Holder (Cuvette): Must be transparent. Glass or clear plastic cuvettes are preferred.

vi. Photo detectors: Detector of colorimeter basically receives the resultant light beam once it has passed through the sample and converts it into electrical signal. Selenium photocell, silicon photocell, phototube, photomultiplier tube etc are used.

vii. Display: It detects and measures the electric signal and makes visible output.

Working of Colorimetry:

i.            Preparation of Sample: The first step involves preparing the sample solution containing the substance of interest. The solution's concentration may be unknown or require quantification.

ii.            Selecting Wavelength: The specific wavelength of light that the absorbing substance interacts with must be chosen. This wavelength is based on the molecule's absorption characteristics. The choice of wavelength affects the accuracy of the measurement.

iii.            Calibration: A series of standard solutions with known concentrations are prepared. These solutions cover a range of concentrations that span the expected concentration of the unknown sample. These standards create a calibration curve that relates absorbance or transmittance to concentration.

iv.            Setting up the Colorimeter: The colorimeter is set up by selecting the appropriate filter to isolate the desired wavelength of light. This ensures that only light of the chosen wavelength reaches the sample.

v.            Blank Measurement: A blank solution, often the solvent used to dissolve the sample, is placed in the cuvette and inserted into the colorimeter. This measurement provides a reference baseline for the instrument to account for any background absorbance or turbidity.

vi.            Sample Measurement: The cuvette containing the sample solution is placed into the colorimeter. Light of the chosen wavelength passes through the solution, and the detector measures the intensity of transmitted light.

vii.            Measurement Comparison: The intensity of transmitted light from the sample is compared to the intensity of transmitted light from the blank solution. The difference in intensity provides the measure of absorbance.

viii.            Calculation: Using the Beer-Lambert law, the absorbance value obtained from the sample measurement is related to the concentration of the substance. The molar absorptive and the path length are constants determined by the specific experimental setup.

ix.            Reference to Calibration Curve: The calculated absorbance value is then referred to the calibration curve created from the standards. By interpolating the absorbance value on the curve, the concentration of the unknown sample is determined.

x.            Reporting Results: The concentration of the unknown sample is reported based on the calibration curve. This concentration value is applicable to the original sample solution.

Applications of Colorimetry:

i.            Quantification of Biological Molecules: Colorimetry plays a pivotal role in the realm of biology by enabling the quantification of essential biological molecules. Techniques such as enzyme assays, DNA quantification, and protein concentration determination heavily rely on colorimetric methods. These methods are prized for their precision and sensitivity, making them indispensable tools for researchers and scientists.

ii.            Enzyme Kinetics and Biochemical Studies: Colorimetric assays are integral to studying enzyme kinetics, unraveling the intricate mechanisms of enzymatic reactions. By measuring changes in color over time, researchers can deduce reaction rates, substrate affinity, and inhibitor effects. These insights contribute to a deeper understanding of cellular processes and enable the development of therapeutic agents.

iii.            Gene Expression Analysis: Colorimetric methods aid in gene expression studies by quantifying nucleic acids. DNA quantification is a fundamental step in molecular biology, and colorimetry provides a straightforward way to assess the amount of DNA in a sample, critical for applications like PCR and cloning.

iv.            Protein-Protein Interactions: Understanding protein interactions is essential in deciphering cellular functions. Colorimetric assays assist in quantifying protein concentrations, enabling the study of protein-protein interactions and their roles in signal transduction, cell communication, and disease mechanisms.

v.            Medical Diagnostics: Colorimetry plays a significant role in medical diagnostics. Blood glucose monitoring relies on color changes to indicate glucose concentrations, aiding diabetes management. Urinalysis employs colorimetry to detect specific compounds indicative of health conditions, simplifying disease diagnosis.

vi.            Environmental Analysis: Beyond the laboratory, colorimetry extends its reach to environmental analysis. It is used to assess water quality, measuring parameters like chemical oxygen demand and pollutant concentrations, contributing to environmental monitoring and conservation efforts.

vii.            Food Quality Assessment: Colorimetry is a key tool in food quality assessment, as color changes in food products can indicate freshness, ripeness, and spoilage. It aids in evaluating color stability and detecting changes during processing and storage.

viii.            Pharmaceutical Testing: In the pharmaceutical industry, colorimetry is employed to determine the concentration of active ingredients in formulations. This ensures the consistency and efficacy of drugs, supporting quality control and regulatory compliance.

Spectrophotometry (Visible, UV and IR):

Spectrophotometry is a technique used to measure the amount of light absorbed or transmitted by a substance as a function of wavelength. It is widely used in various scientific fields, including chemistry, biochemistry, physics, and environmental science, for quantitative analysis of substances that absorb light. Spectrophotometry is based on the fundamental principle of the interaction between electromagnetic radiation (light) and matter.   

The basic principle of spectrophotometry involves the interaction between light and a sample. When light passes through a sample, some of it may be absorbed by the sample's molecules. The amount of light absorbed is related to the concentration of the absorbing species in the sample. By measuring the intensity of light before and after it passes through the sample, we can determine the extent of absorption and, consequently, the concentration of the absorbing substance.

There are three main types of spectrophotometry based on the regions of the electromagnetic spectrum they utilize:

Visible Spectrophotometry: This type of spectrophotometry uses visible light, which spans the wavelength range of approximately 400 to 700 nanometers (nm). Visible spectrophotometry is commonly used for analyzing colored compounds, as the colors we perceive are a result of selective absorption of light by certain molecules. This technique is particularly useful for analyzing transition metal complexes, organic dyes, and various chromophores.

UV (Ultraviolet) Spectrophotometry: UV spectrophotometry operates in the ultraviolet range of the electromagnetic spectrum, typically between 200 and 400 nm. UV spectroscopy is valuable for studying molecules that contain conjugated double bonds, aromatic rings, and functional groups that exhibit absorption in this region. It's often used to analyze nucleic acids, proteins, and certain organic compounds.

IR (Infrared) Spectrophotometry: IR spectrophotometry is based on the interaction of infrared radiation with a sample. The IR region spans from about 4000 to 400 cm^-1 (wavenumbers) or 2.5 to 25 micrometers (μm). IR spectroscopy provides information about the vibrational modes of molecules, allowing for the analysis of functional groups and molecular structures. It is commonly used for identifying organic and inorganic compounds, as well as for characterizing polymers and determining the presence of specific chemical bonds.

Principle of Spectrophotometry: The principle of spectrophotometry is based on the interaction between electromagnetic radiation (light) and matter. When light passes through a sample, some of it may be absorbed by the molecules in the sample. The amount of light absorbed is directly proportional to the concentration of the absorbing species in the sample. This relationship is described by the Beer-Lambert Law, which is the fundamental equation that governs spectrophotometric measurements:

The Beer-Lambert Law states that the absorbance of a sample is directly proportional to the concentration of the absorbing species and the path length, and it's also influenced by the molar absorptivity of the substance at a specific wavelength.

Instrumentation of Spectrophotometry: Spectrophotometers are the instruments used to perform spectrophotometric measurements. They consist of several key components that work together to measure the absorbance or transmittance of light through a sample.

Here's an overview of the basic components:

1.      Light Source: A light source emits a beam of light with a broad range of wavelengths, typically covering the UV, visible, or IR regions of the electromagnetic spectrum. Common light sources include tungsten lamps, deuterium lamps (for UV), and halogen lamps.

2.      Monochromator: The monochromator is a critical component that selects a specific wavelength from the light emitted by the source. It consists of a prism or diffraction grating that disperses the incoming light into its individual wavelengths. By adjusting the position of the prism or grating, a specific wavelength is isolated and directed towards the sample.

3.      Sample Compartment: This is where the sample is placed for analysis. It typically consists of a cuvette or sample cell made of transparent material (usually glass or quartz) that allows light to pass through. The cuvette holds the sample solution, and its dimensions (path length) are important for accurate measurements.

4.      Detector: The detector measures the intensity of light that exits the sample. Common detectors include photodiodes, photomultiplier tubes (PMTs), and charge-coupled devices (CCDs). The detector converts the light signal into an electrical signal that can be processed and analyzed.

5.      Amplifier and Signal Processing: The electrical signal from the detector is often weak, so an amplifier is used to increase its strength. Signal processing electronics then convert the amplified signal into a digital format for further analysis.

6.      Display and Data Output: The processed data can be displayed on a screen as an absorbance or transmittance value. Data can also be output to a computer for further analysis and storage.

Working of Spectrophotometry: The working of spectrophotometry involves the interaction of light with a sample and the subsequent measurement of the absorbed or transmitted light. Here's a step-by-step overview:

Ø  The light source emits a beam of light with a range of wavelengths. This light passes through the monochromator.

Ø  The monochromator selects a specific wavelength of light by either refracting it through a prism or diffracting it using a grating. The monochromator allows only the selected wavelength to pass through.

Ø  The monochromatic light beam enters the sample compartment where the sample solution is located in a cuvette.

Ø  As the monochromatic light passes through the sample, some of it is absorbed by the molecules in the sample. The remaining light is transmitted through the sample.

Ø  The detector measures the intensity of the transmitted light. If the sample absorbs a significant amount of light, the detector will record a lower intensity.

Ø  The detector's signal is amplified and processed electronically to convert it into a digital format.

Ø  The absorbance (A) is calculated using the formula A = -log(T), where T is the transmittance of light through the sample.

Ø  The absorbance value can be correlated with the concentration of the absorbing species in the sample using the Beer-Lambert Law. This allows for quantitative analysis of the sample's components.

Ø  The calculated data can be displayed on the instrument's screen or output to a computer for further analysis and interpretation.

Applications of Spectrophotometry: Spectrophotometry is a versatile analytical technique with a wide range of applications across various scientific fields. It is particularly valuable for its ability to quantitatively analyze the concentration of absorbing or transmitting substances in a sample. Here are some key applications of spectrophotometry:

1.                  Quantitative Analysis: Spectrophotometry is widely used for quantitative analysis of chemical compounds. By measuring the absorbance or transmittance of light through a sample, the concentration of the target substance can be determined using the Beer-Lambert Law. This is applied in areas such as pharmaceuticals, environmental monitoring, and clinical diagnostics.

2.                  Biochemical Analysis: In biochemistry, spectrophotometry is used to quantify biomolecules such as proteins, nucleic acids (DNA, RNA), and enzymes. Different biomolecules absorb light at specific wavelengths, allowing researchers to measure their concentrations and study their properties.

3.                  Pharmaceutical Analysis: Spectrophotometry is crucial in pharmaceutical quality control to ensure the purity and concentration of active pharmaceutical ingredients (APIs) in drugs. It's also used to monitor dissolution profiles, stability, and degradation of drug formulations.

4.                  Environmental Monitoring: Spectrophotometry is used to analyze pollutants, nutrients, and contaminants in environmental samples such as water, air, and soil. It helps monitor water quality, detect harmful substances, and assess the impact of human activities on ecosystems.

5.                  Food and Beverage Industry: The analysis of food and beverages often involves spectrophotometry to determine characteristics like color, flavor compounds, preservatives, antioxidants, and nutrient content. It's used in quality control and product development.

6.                  Clinical Diagnostics: In clinical laboratories, spectrophotometry is used to measure various biomarkers in blood and urine samples. It's employed in tests such as blood glucose, cholesterol, hemoglobin, and liver function tests.

7.                  DNA and RNA Quantification: Spectrophotometry is commonly used to quantify DNA and RNA concentrations in molecular biology research. This is important for tasks like PCR (polymerase chain reaction), DNA sequencing, and gene expression studies.

8.                  Drug Development: It plays a role in drug development by assessing the interactions between drugs and target molecules. It's used to determine binding constants, affinity, and kinetics of drug-receptor interactions.

9.                  Forensic Analysis: It is used in forensic science to analyze trace evidence, such as detecting and quantifying drugs, toxins, and chemicals in crime scene samples.

10.              Material Analysis: It can be used to analyze the optical properties of materials, such as determining the bandgap of semiconductors or studying the absorption characteristics of pigments and dyes.

CHROMATOGRAPHY:

Chromatography is a collective term for a family of laboratory techniques used to separate and analyze mixtures of compounds into their individual components. The fundamental principle of chromatography is based on the differential interaction of components between a mobile phase (a fluid that carries the sample) and a stationary phase (a solid or liquid material that interacts with the sample).

Chromatography is a technique to separate and analyze mixtures.

It works like in following basic manner:

a.      Setup: A mixture is placed on a material (stationary phase).

b.      Flow: A liquid (mobile phase) carries the mixture through the material.

c.       Separation: Different parts of the mixture interact differently with the material and mobile phase, causing them to move at various speeds.

d.      Result: The mixture gets divided into its components.

Chromatography helps scientists identify, analyze, and purify substances. It's used in labs, industries, and research.

The key types of chromatography include:

1.      Thin-Layer Chromatography (TLC): In TLC, a thin layer of stationary phase (usually silica gel or a similar material) is coated onto a flat support (typically a glass plate). The sample is applied as a spot at one end, and the plate is then developed in a solvent. Compounds move up the plate at different rates based on their affinities for the stationary and mobile phases.

2.      Gas Chromatography (GC): GC is used to separate volatile compounds. The stationary phase is a liquid coated onto an inert solid support inside a long, coiled column. The sample is vaporized and introduced into the column using an inert carrier gas. Compounds separate based on their volatilities and interactions with the stationary phase.

3.      Liquid Chromatography (LC): In LC, the stationary phase is a liquid, often immobilized on a solid support. High-Performance Liquid Chromatography (HPLC) is a widely used form of LC that utilizes high pressure to achieve faster and more efficient separations.

4.      Affinity Chromatography: In this technique, the stationary phase is designed to have a specific affinity for a target compound or class of compounds. It's commonly used to purify proteins and other biomolecules.

5.      Column Chromatography: As described in the previous response, this involves passing a sample mixture through a column filled with a stationary phase, where compounds separate based on their interactions with the stationary and mobile phases.

6.      Ion-Exchange Chromatography: This method separates compounds based on their ionic charges. The stationary phase contains charged groups that attract or repel ions in the sample.  It is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It can be used for almost any kind of charged molecules including large protein, small nucleotide and amino acids. The solution to be injected is called Sample and individually separated components are called analytes.

7.      Size-Exclusion Chromatography (SEC) or Molecular sieve chromatography: Also known as gel filtration chromatography, SEC separates molecules based on their size and shape. The stationary phase is a porous material that allows smaller molecules to enter its pores, causing them to take longer to traverse the column. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers.

General account of Column chromatography,

Column chromatography is a separation technique used in chemistry to separate and purify mixtures of compounds based on their interactions with a stationary phase and a mobile phase.

Here's an overview of how column chromatography works:

Principle: Column chromatography relies on the fact that different compounds have varying affinities for a stationary phase (solid material) and a mobile phase (liquid solvent). Compounds with stronger interactions with the stationary phase will move more slowly through the column, while those with weaker interactions will move more quickly.

Setup:

v  Column: A glass or plastic column is filled with a stationary phase, often a fine-grained material like silica gel or alumina. The stationary phase provides surface area for interactions.

v  Packing: The stationary phase is packed uniformly to avoid preferential flow paths and ensure efficient separation.

v  Sample Application: The mixture to be separated is dissolved in a suitable solvent and carefully applied to the top of the column.

Separation:

v  Elution: The mobile phase, a solvent, is added to the column's top. It flows down through the stationary phase, carrying the sample compounds with it.

v  Interactions: Compounds interact differently with the stationary phase. Some might bind more strongly and move more slowly, while others bind less and move faster.

v  Retention Times: The time it takes for each compound to pass through the column and elute at the bottom depends on its interactions with the phases.

Collection:

v  Fractions: As compounds elute from the column, they're collected in separate fractions based on their elution times.

v  Analysis: Fractions can be analyzed further using techniques like thin-layer chromatography, spectroscopy, or mass spectrometry to identify and quantify the separated compounds.

Applications:

v  Column chromatography is versatile and can handle larger sample sizes compared to thin-layer chromatography.

v  Proper choice of stationary and mobile phases is crucial for successful separation.

v  Gradient elution (changing mobile phase composition) can enhance separation efficiency.

v  Preparative column chromatography can provide purified compounds for research or applications.

Ø Adsorption Chromatography:

Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.

Principle: Adsorption chromatography is based on the concept of compounds in a sample mixture interacting differently with a solid stationary phase. The stronger the interaction between a compound and the stationary phase's surface, the longer the compound will take to move through the column. Weaker interactions lead to faster elution.

Type of Adsorption Chromatography:

i)                    Column Chromatography,

ii)                  Thin Layer Chromatography,

iii)                Gas Solid Chromatography Types

Stationary Phase or Bedding Materials for Adsorption Chromatography:

a)      Silica Gel (SiO2): Silica gel is a popular choice for adsorption chromatography. It consists of porous silica particles that provide a large surface area for adsorption interactions. Compounds in the mixture can form hydrogen bonds, van der Waals forces, or other interactions with the surface of the silica gel.

b)      Alumina (Al2O3): Similar to silica gel, alumina is another common adsorbent used as a stationary phase. It also offers a high surface area for interactions with sample components.

c)      Cellulose: In thin-layer chromatography, cellulose is coated onto a solid support, forming a thin layer for separation. It's commonly used for separating compounds based on their polarities.

Ø Partition Chromatography:

This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

Principle: Partition chromatography operates on the principle of different compounds partitioning or distributing themselves between two immiscible liquid phases—the stationary liquid phase immobilized on a solid support and the mobile liquid phase (solvent). The stationary liquid is usually more polar than the mobile phase.

Partition of component of sample between sample and liquid/ gas stationary phase retard some components of sample more as compared to others. This gives basis for separation.

The stationary phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phase passes over the coated adsorbent and depending upon relative solubility in the coated liquid, separation occurs. The component of sample mixture appear separated because of differences in their partition coefficient.

Partition Chromatography types

i)                    Liquid-liquid Chromatography

ii)                  Gas-liquid Chromatography

Stationary Phase or Bedding Materials of Partition chromatography:

a)      Solid Support: The solid support provides stability and structure to the stationary phase. Common materials include glass beads, silica particles, and other inert materials.

b)      Liquid Phase: The liquid stationary phase is coated onto the solid support. Commonly used liquids include organic solvents that form a thin film on the solid surface. The choice of liquid depends on the types of compounds being separated.

In both types of chromatography, the stationary phase's properties and interactions with the sample components are crucial for separation. The choice of stationary phase material impacts the separation efficiency and selectivity of the chromatographic technique. By exploiting these interactions, chromatography allows scientists to separate and analyze complex mixtures effectively.

Ø Ion exchange chromatography,

Ion Exchange Chromatography (Ion Chromatography) is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It can be used for almost any kind of charged molecules including large protein, small nucleotide and amino acids. The solution to be injected is called Sample and individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.

Principle:

Ion Exchange Chromatography is based on the relative retention of the ions during their progress through an ion exchange column which has functional group of opposite charge attached to its surface. The stronger the charge on the ion, the greater is the retention time in the column. Ion chromatography is used to separate organic or inorganic charged substances. The stationary phases used are based on typical ion exchange resins.

Stationary Phase Materials:

The stationary phase in ion exchange chromatography is typically composed of ion exchange resins. These resins are porous polymer beads that have covalently attached charged groups. The choice of resin depends on whether you're dealing with cations (positively charged ions) or anions (negatively charged ions) and the desired separation conditions.

There are two main types of ion exchange resins:

i)                    Cation Exchange Resins: These resins have negatively charged functional groups (such as sulfonic acid groups). They attract and bind positively charged ions (cations) from the sample. Common cations include sodium (Na+), potassium (K+), calcium (Ca2+), and others.

ii)                  Anion Exchange Resins: These resins have positively charged functional groups (such as quaternary ammonium groups). They attract and bind negatively charged ions (anions) from the sample. Common anions include chloride (Cl-), sulfate (SO42-), and others.

Working:

Sample Loading: The mixture containing ions is applied to the ion exchange column, which contains the resin.

i)                    Ion Exchange: As the sample passes through the column, ions in the mixture interact with the charged groups on the resin. Ions that have a higher affinity for the resin will bind more tightly and elute later, while ions with lower affinity will elute earlier.

ii)                  Elution: To separate the ions, a gradient of increasing ionic strength or a change in pH is typically applied. This weakens the interactions between the ions and the resin, causing them to be displaced and eluted from the column at different times.

Applications:

Ion exchange chromatography is widely used in various fields, including biotechnology, pharmaceuticals, environmental analysis, and water purification. It's used for tasks such as purifying proteins, separating ions from complex mixtures, and preparing samples for further analysis.

Ø Molecular sieve chromatography (Size-Exclusion Chromatography):

Molecular sieve chromatography, also known as size-exclusion chromatography (SEC), separates molecules based on their size and shape. The stationary phase consists of porous beads with different pore sizes. Larger molecules cannot enter the pores and are excluded from them, resulting in faster elution. Smaller molecules can enter the pores and are retained longer, leading to slower elution.

Stationary Phase Material:

The stationary phase in molecular sieve chromatography is typically composed of porous beads made from materials like cross-linked agarose or polyacrylamide. These beads have pores of various sizes, creating a range of exclusion limits for different-sized molecules.

Working:

i)                    Sample Loading: The mixture containing molecules of different sizes is applied to the column packed with the stationary phase beads.

ii)                  Exclusion Effect: Larger molecules cannot enter the smaller pores and are excluded from them. They flow around the beads and are eluted more quickly.

iii)                Retention: Smaller molecules can enter the pores and are temporarily trapped within the beads. This results in their slower elution.

iv)                Elution: As the solvent continues to flow through the column, molecules are eluted in order of their size, with the largest molecules eluting first and the smaller ones eluting later.

Applications:

i)                    Protein and Polymer Separation: It's used to separate proteins, polymers, and other biomolecules based on their molecular sizes.

ii)                  Purification: It's employed to remove unwanted high-molecular-weight impurities from samples.

iii)                Quality Control: Molecular sieve chromatography is used in quality control of products in industries like pharmaceuticals and biotechnology.

iv)                Environmental Analysis: It's used to analyze and characterize natural and synthetic polymers in environmental samples.

v)                  Polymer Research: Molecular sieve chromatography is crucial for studying polymer distributions and molecular weights.

vi)                Biochemical Research: It's used to analyze protein aggregates and study macromolecular interactions.

vii)              Biotechnology: It's employed in the purification and characterization of biopharmaceuticals.

UNIT IV 

PHARMACOGNOSY AND MEDICINAL BOTANY

Monographs of drugs with reference to biological sources, geographical distribution, common varieties, macro and microscopic characters, chemical constituents, therapeutic uses, adulterants- in

1.      Strychnos seeds,

2.      Senna leaves,

3.      Clove buds,

4.      Allium sativum,

5.      Acorus calamus and

6.      Curcuma longa

1.      Strychnos Nux Vomica

Biological sources: Strychnos Nux Vomica, a fascinating botanical specimen, belongs to the Loganiaceae family. This plant is also known by several intriguing names, including "poison nut," "semen strychnos," "kuchla," and "quaker buttons." However, its true intrigue lies in the seeds that it bears.

Geographical Distribution: It is mainly found in South India, Malabar Coast, Kerala, Bengal, Eastern Ghats, North Australia and Ceylon.

Morphological/ Macroscopical features:

• It is a deciduous tree and medium sized tree. It have short, crooked, thick trunk and the wood is white and hard.

• Branches are irregular and covered with a smooth ash- coloured bark.

• Leaves are opposite, short stalked.

• The shape of leaves is oval and smooth and shiny on both sides.

• The size of leaves is about 4 inches long and 3 inches broad.

• Flowers are small, funnel shape in small terminal cymes .Color of flowers are greenish-white.

• Fruit contain smooth hard rind or shell and ripe have orange color and have jelly like pulp.

• Fruit have five seeds covered with a soft woolly like substance which is white and horny internally.

• The shape of seed is flattened disks densely covered with closely appressed satiny hairs.

• The size of seed is 10-30 mm in diameter and 3-5 mm thick, radiating from the centre of the flattened sides which give a characteristic sheen to the seeds.

• Seeds contain dark grey horny endosperm in which the small embryo is embedded.

• It have no odour but a very bitter taste.

Microscopical Character: Epidermis consists of thick-waved, bent and twisted lignified covering trichomes. The base of the trichome is large thick walled with slit like pits. The upper part of the trichome is nearly at right angle to the base and has wavy walls. Endosperm consists of thick walled isodiametric cells consisting of hemicellulose which swells with water and contains plasmodesma. Aleurone grains and fixed oil are present in endosperm and embryo.

Chemical constituents :

• Alkaloids are the major chemical constituent of nux vomica.

• It contain 1.25% strychnine and 1.5% brucine.  It contain glucoside loganin and about 3% fatty matter.  It contain caffeotannic acid and trace of copper. Nux-vomica consist of 2.5-3.5 % bitter indole alkaloids.

• Strychnine is the major chemical constituent of nux vomica which is therapeutically active and toxic alkaloid which is located in the central portion of endosperm.

• The another major constituent of nux vomica is brucine which is chemically dimethoxystrychnine and it is less toxic which have very low physiological action.

• It is intensely bitter and used as a standard determining the bitter value, of many bitter drugs.

Hemicelluloses is a reserved material found in the thick cell wall of endosperm.

Chemical Tests

1. Strychnine Test: To a section of endosperm add ammonium vanadate and sulphuric acid. Strychnine in the middle portion of endosperm is stained purple.

2. Potassium dichromate test: Strychnine gives violet colour with potassium dichromate and conc. sulphuric acid.

3. Brucine Test: To a thick section add concentrated nitric acid. Outer part of endosperm is stained yellow to orange because of brucine.

4. Hemicellulose Test: To a thick section add iodine and sulphuric acid. The cell walls are stained blue.

Therapeutic uses:

• It is used to increase appetite i.e. it stimulates peristalsis in chronic constipation due to atony of the bowel it is often combined with cascara and other laxatives with good effects.

• Strychnine is the major chemical constituent of nux vomica helps to increase the flow of gastric juice.

• The major constituent of nux vomica, strychnine has a stimulant action on spinal cord and reflex movements are better.

• It is used to treat cardiac failure because smell, touch, hearing and vision are rendered more acute and improves pulse and rise blood pressure.

• The other chemical constituent of nux vomica is brucine which has less poisonous nature and used in pruritis.

• It is also used as a local anodyne in inflammations of the external ear.

• It is used to treat neurasthenia ,a diseased condition in which excessive fatigue occur in neurotic origin.

• It is a bitter stomachic, which strengthen stomach and promote its action.

Adulterants : The common adulterants of nux vomica are Strychnine potatorum and Strychnine nux blanda.  Strychnine potatorum commonly known as clearing nut which do not contain alkaloids ,even in small amount and also it is not bitter.

2.      Senna leaves

Biological Source: Senna leaf consists of the dried leaflets of Cassia acutifolia Delile (C. senna L.) known as Alexandrian senna and of C. angustifolia Vahl., which is commercially known as Tin-nevelly senna. It belong family Leguminosae.

Geographical Source: Alexandrian senna is indigenous to South Africa. It widely grows and sometimes is cultivated in Egypt and in the middle upper territories of Nile river. It is also cultivated in Kordofan and Sennar regions of Sudan. Indian or Tinnevelly senna is indigenous to southern Arabia and cultivated largely in Tinnevelly and Ramnathpuram districts of Tamilnadu. It also grows in Somaliland, Sindh and Punjab region.

Macroscopic / Morphological characters: Senna leaflets are 3–5 cm long, 2 cm wide and about 0.5 mm thick. It shows acute apex, entire margin and asymmetric base. Outline is lanceolate to ovate lanceolate. Pubescent lamina is found on both the surfaces. Leaves show greyish green colour for Alexandrian senna and yellowish green for Tinnevelly senna.

Leaves of Tinnevelly senna are somewhat larger, less broken and firmer in texture than that of Alexandrian senna. Odour of leaves is slight but characteristic and the taste is bitter, mucilagenous. Both the types of leaflets show impression or transverse markings due to the pressing of midrib.

Distinguishing characters of Alexandrian and Indian senna are given in Table below.

Microscopical characters: Being isobilateral leaf, senna shows more or less similar features at both the surfaces of leaf with few differences. Transverse section of leaf shows upper and lower epidermis with straight wall cells, few of which contain mucilage. Paracytic stomata and nonlignified unicellular trichomes are found on both the surfaces. A single layer of palisade parenchyma is observed at both the sides but it is discontinued in the midrib region of lower epidermis due to the zone of collenchymatous tissues. Palisade is followed by spongy mesophyll which contains cluster crystals of calcium oxalate and vascular strands. Midrib shows the vascular bundle containing xylem and phloem, almost surrounded by lignified pericyclic fibres and a sheath of parenchyma which contains prismatic crystals of calcium oxalate.

Chemical Constituents : Senna contains sennosides A and B (2.5%) based on the aglycones sennidin A and B, sennosides C and D which are glycosides of heterodianthrones of aloe-emodin and rhein are present. Others include palmidin A, rhein anthrone and aloe-emodin glycosides. Senna also contains free chryso phanol, emodin and their glycosides and free aloe-emodin, rhein, their monoanthrones, dianthrones and their glycosides. Mucilage is present in the epidermis of the leaf and gives red colour with ruthenium red.

Chemical Test

Borntrager test for anthraquinones: The leaves are boiled with dilute sulphuric acid and filtered. To the filtrate organic solvent like benzene, ether or chloroform is added and shaken. The organic layer is separated, and to it add ammonia solution. The ammoniacal layer produces pink to red colour indicating the presence of anthraquinone glycoside.

Therapeutic uses: Senna leaves are used as laxative. It causes irritation of large intestine and have some griping effect. Thus they are prescribed along with carminatives. Senna is stimulant cathartic and exerts its action by increasing the tone of the smooth muscles in large intestine.

Adulterants

Cassia obovata (Dog Senna): They occur as small pieces with Alexandrian senna but can be easily identified by its obovata shape and obtuse and tapering apex. It has only 1% anthraquinone derivatives. The presence of Cassia auriculata (Palthe senna) can be identified by treating it with 80% sulphuric acid. It gives red colour.

Cassia marilandica or American Senna, Wild Senna, Poinciana pulcherima, formerly Maryland Senna, is a common perennial from New England to Northern Carolina. Its leaves are compressed into oblong cakes like other herbal preparations of the Shakers. It acts like Senna, but is weaker, and should be combined with aromatics. These leaves are also found mixed with or substituted for Alexandrian Senna.

Cassia montana yields a false Senna from Madras, partly resembling the Tinnevelly Senna, though the colour of the upper surface of the leaves is browner.

3.      Clove Bud

Biological Source: Clove consists of the dried flower buds of Eugenia caryophyllus Thumb., belonging to family Myrtaceae.

Geographical Source: Clove tree is a native of Indonesia. It is cultivated mainly in Islands of Zanzibar, Pemba, Brazil, Amboiana, and Sumatra. It is also found in Madagascar, Penang, Mauritius, West Indies, India, and Ceylon.

Macroscopical/Morphological Characters: Clove is reddish-brown in colour, with an upper crown and a hypanthium. The hypanthium is sub-cylindrical and tapering at the end. The hypanthium is 10 to 13 mm long, 4 mm wide, and 2 mm thick and has schizolysigenous oil glands and an ovary which is bilocular. The Crown region consists of the calyx, corolla, style and stamens. Calyx has four thick sepals. Corolla is also known as head, crown or cap; it is doineshaped and has four pale yellow coloured petals which are imbricate, immature, and membranous. The ovary consists of abundant ovules. Clove has strong spicy, aromatic odour, and pungent and aromatic taste.

Microscopical Character: The transverse section should be taken through the short upper portion which has the bilocular ovary and also through the hypanthium region. The transverse section through the hypanthium shows the following characters. It has a single layer of epidermis covered with thick cuticle.

The epidermis has ranunculaceous stomata. The cortex has three distinct region: the peripheral region with two to three layers of schizolysigenous oil glands, embedded in parenchymatous cells. The middle layer has few layers of bicollateral vascular bundle. In the inner portion it has loosely arranged aerenchyma cells. The central cylinder contains thick-walled parenchyma with a ring of bicollateral vascular bundles and abundant sphaeraphides. The T.S. through ovary region shows the presence of an ovary with numerous ovules in it.

Chemical Constituents: Clove contains 14–21% of volatile oil. The other constituents present are the eugenol, acetyl eugenol, gallotannic acid, and two crystalline principles; α- and β- caryophyllenes, methyl furfural, gum, resin, and fibre. Caryophyllin is odourless component and appears to be a phytosterol, whereas eugenol is a colourless liquid. Clove oil has 60–90% eugenol, which is the cause of its anesthetic and antiseptic properties.

Chemical Tests

1. To a thick section through hypanthium of clove add 50% potassium hydroxide solution; it produces needle-shaped crystals of potassium eugenate.

2. A drop of clove oil is dissolved in 5 ml alcohol and a drop of ferric chloride solution is added; due to the phenolic OH group of eugenol, a blue colour is seen.

3. To a drop of chloroform extract of clove add a drop of 30% aqueous solution of sodium hydroxide saturated with sodium bromide; Needle and pear shaped crystals of sodium eugenate arranged in rosette are produced immediately.

Therapeutic uses: Clove is used as an antiseptic, stimulant, carminative, aromatic, and as a flavouring agent. It is also used as anodyne, antiemetic. Dentists use clove oil as an oral anesthetic and to disinfect the root canals. Clove kills intestinal parasites and exhibits broad antimicrobial properties against fungi and bacteria and so it is used in the treatment of diarrhea, intestinal worms, and other digestive ailments. Clove oil can stop toothache. A few drops of the oil in water will stop vomiting, eating cloves is said to be aphrodisiac. Eugenol is also used as local anaesthetic in small doses. The oil stimulates peristalsis; it is a strong germicide, also a stimulating expectorant in bronchial problems. The infusion and Clove water are good vehicles for alkalies and aromatics.

Adulterants: The clove is generally adulterated by exhausted clove, clove fruits, blown cloves and clove stalks. The exhausted cloves are those from which volatile oil is either partially or completely removed by distillation. Exhausted cloves are darker in colour and can be identified as they float on freshly boiled and cooled water. Clove fruits are dark brown in colour and have less volatile oil content. These can be identified by the presence of starch present in the seed of the fruit. Blown Cloves are entirely developed clove flowers from which corolla and stamens get separated. While sepa-ration, sometimes the stalks are incompletely removed and the percentage of volatile oil in clove stalk is only 5%. As clove stalks contain prism type of calcium oxalate crystals and thick-walled stone cells which are absent in clove the clove stalk can also be detected.

4.      Allium sativum

Biological Source: Garlic is the ripe bulb of Allium sativum Linn., belonging to family Liliaceae.

Geographical Source: Garlic occurs in central Asia, southern Europe, and United States. It is widely cultivated in India.

Macro/ Morphological Characteristics: Garlic is bulbous perennial herb. It has tall, erect flowering stem that reaches 2-3 feet in height. The plant has pink or purple flowers that bloom in mid to late summer. The part used medicinally is the bulb. Bulb 4-6 cm in diameter, consisting of 8-20 cloves (bulblets), surrounded by 3-5 whitish papery membranous scales attached to a short, disc-like woody stem having numerous, wiry rootlets on the underside; each clove is irregularly ovoid, tapering at upper end with dorsal convex surface, 2-3 cm long, 0.5 - 0.8 cm wide, each surrounded by two thin papery whitish and brittle scales having 2-3 yellowish green folded leaves contained within two white fleshy, modified leaf bases or scales; odour is Characteristic, taste is pungent gives warmth to the tongue  It is a perennial herb having bulbs with several cloves, enclosed in a silky white or pink membraneous envelope.

Microscopical characteristics:  The transverse section of bulb of Allium sativum has cuticle, epidermis, cortex, endodermis and scattered vascular bundles. Epidermis consists of narrow thin walled continuous single layered with rectangular cells, surrounded by cuticle. The cortex region have parenchymatous and homogenous cell having large prismatic crystals of calcium oxalate and vascular bundles.  The vascular bundles are bicollateral small, occur as a ring and scattered in the median part of cortex and circularly arranged below endodermis.

Chemical Constituents: Allicin, a yellow liquid responsible for the odour of garlic, is the active principle of the drug. It is miscible with alcohol, ether, and benzene and decomposes on distilling. The other constituents reported in Garlic are alliin, volatile and fatty oils, mucilage and albumin. Alliin, another active principle, is odourless, crystallized from water acetone and practically insoluble in absolute alcohol, chloroform, acetone, ether, and benzene. The amino acids present in the bulb are leucine, methionine, S-propyl-L-cysteine, S-propenyl-L-cysteine, S-methyl cysteine, S-allyl cysteine sulphoxide (alliin), S-ethyl cysteine sulphoxide, and S-butyl-cysteine sulphoxide.

Therapeutic uses: Garlic is carminative, aphrodisiac, expectorant, stimulant, and used in fevers, coughs, febrifuge in intermittent fevers, respiratory diseases such as chronic bronchitis, bronchial asthma, whooping cough, and tuberculosis. It is also used in atherosclerosis and hypertension.

Garlic is consumed as a complement in the diet of hyperlipidemic patients and for the prophylaxis of the vascular changes induced by ageing. The garlic can cause gastrointestinal distress and alters breath and skin odour. Garlic or its constituents exhibit various biological activities, such as antibacterial, antifungal, antiviral, antitumor, and ant diabetic effects.

5.      Acorus calamus

Biological Source: Calamus consists of dried rhizomes of Acorus calamus Linn., (Araceae).

Geographical distribution: Grows in India, central Asia, southern Russia and Siberia, and Europe.Habitats include edges of small lakes, ponds and rivers, marshes, swamps, and wetlands

Morphology/Macroscopical characters: It is a semiaquatic perennial plant. The plant grows from 60 to 100 cm tall. The stem is triangular and sprouts from a horizontal, round rootstock, which has the thickness of a thumb. The leaves are yellowish-green, 2 to 3 feet in length, oblong, sword-shaped, tapering into a long, acute point, often undulate on the margins and arranged in two rows.

The rhizome has an intensely aromatic fragrance and a tangy, pungent and bitter taste. The flowers are small dice-shaped, slim, conical spadix, greenish in colour appear from May to July. Fruits are berries, full of mucus, which falls when ripe into the water or to the ground. Rhizomes are about 20 cm long, 1 to 2 cm in diameter, either peeled or unpeeled, reddish grey in colour, soft, porous, with longitudinal furrows. On the lower surface there are small root scars which are slightly raised.

Microscopic characters: Transverse section is differentiated into narrow cortical and large stelar regions. Epidermis is single layered having radially elongated cells with heavily thickened outer walls: Cortical region consists of thin walled parenchymatous cells arranged in chains leaving large intercellular spaces, sheathed collateral vascular bundles and bundles of fibers. Stelar region is outlined by single layer of barrel-shaped endodermal cells with abundant starch grains. Mostly leptocentrics and few collateral vascular bundles in association with the leptocentrics are observed in the ground tissue of the stele. Vessels are with simple and scalariform pits. Fibers arc thick-walled and pitted. Large oil cells, dark brown oleoresin content and starch grains are scattered in the ground tissue of both the cortex and stele.

Chemical Constituents: The dried rhizome contains about 1.5–2.7% of a neutral, yellow, aromatic, essential oil. The fresh aerial parts yield about 0.12% of the volatile oil, whereas the unpeeled roots yield the maximum of 1.5–3.5%. The constituents present in calamus are acorin a volatile essential oil, amorphous, which is semifluid, resinous, neutral in reaction, bitter and aromatic, and soluble in alcohol, chloroform and ether; acoretin or choline is a bitter principle with resinous nature; a crystalline alkaloid soluble in alcohol and chloroform, Calamine; along with other constituents like bitter glucoside, starch, mucilage and traces of tannin. The volatile oil is yellowish-brown in colour and is composed of asaryl aldehyde, heptylic and palmitic acid, eugenol, esters of acetic and palmitic acids, pinene, camphene, sesquiterpene, calamene and a small quantity of phenol, methyl eugenol, cilamenenol, and calameone.

Therapeutic uses: Calamus is an aromatic, bitter stomachic, carminative, appe-tizer, digestive, spasmolytic, stomach tonic, nervine sedative, and antiperiodic. The volatile oil is aromatic, expectorant and antiseptic, as a flavouring agent, in perfumery. The dried root and rhizomes are chewed to relieve dyspepsia, bronchitis and also chewed to clear the voice.

6.      Curcuma longa

Biological Source: Turmeric is the dried rhizome of Curcuma longa Linn. (Zingiberaceae).

Geographical distribution: The plant is a native to southern Asia and is cultivated extensively in temperate regions. It is grown on a larger scale in India, China, East Indies, Pakistan, and Malaya.

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Macro/ Morphological Characteristics: The primary rhizomes are ovate or pear-shaped, oblong or pyriform or cylindrical, and often short branched. The rhizomes are known as ‘bulb’ or ‘round’ turmeric. The sec-ondary, more cylindrical, lateral branched, tapering on both ends, rhizomes are 4–7 cm long and 1–1.5 cm wide and called as ‘fingers’.

The bulbous and finger-shaped parts are separated and the long fingers are broken into convenient bits. They are freed from adhering dirt and fibrous roots and subjected to curing and polishing process. The curing consists of cooking the rhizomes along with few leaves in water until they become soft. The cooked rhizomes are cooled, dried in open air with intermittent turning over, and rubbed on a rough surface. Colour is deep yellow to orange, with root scar and encircling ridge-like rings or annulations, the latter from the scar of leaf base. Fracture is horny and the cut surface is waxy and resinous in appearance. Outer surface is deep yellow to brown and longitudinally wrinkled. Taste is aromatic, pungent and bitter; odour is distinct.

Microscopic characters: The transverse section of the rhizome is characterized by the presence of mostly thin-walled rounded parenchyma cells, scattered vascular bundles, definite endodermis, few layers of cork developed under the epidermis, and scattered oleoresin cells with brownish contents. The epidermis is consisted of thick-walled cells, cubical in shape, of various dimensions. The cork cambium is developed from the sub-epidermal layers and even after the development of the cork, the epidermis is retained. Cork is generally composed of four to six layers of thin-walled brick-shaped parenchymatous cells. The parenchyma of the pith and cortex contains grains altered to a paste, in which sometimes long lens shaped unaltered starch grains of 4–15 μm diameter are found. Oil cells have suberised walls and contain either orange-yellow globules of a volatile oil or amorphous resinous masses. Cortical vascular bundles are scattered and are of a collateral type. The vascular bundles in the pith region are mostly scattered and they form discontinuous ring just under the endodermis. The vessels have mainly spiral thickenings and only a few have reticulate and annular structure.

Chemical constituents: Turmeric contains yellow colouring matter called as curcuminoids (5%) and essential oil (6%). The chief constituent of the colouring matter is curcumin I (60%) in addition with small quantities of curcumin III, curcumin II and dihydrocurcumin. The volatile oil contains mono- and sesquiterpenes like zingiberene (25%), α-phellandrene, sabinene, turmerone, arturmerone, borneol, and cineole. Choleretic action of the essential oil is attributed to β-tolylmethyl carbinol.

Chemical Tests

1. Turmeric powder on treatment with concentrated sulphuric acid forms red colour.

2. On addition of alkali solution to Turmeric powder red to violet colour is produced.

3. With acetic anhydride and concentrated sulphuric acid Turmeric gives violet colour. Under UV light this colour is seen as an intense red fluorescence.

4. A paper containing Turmeric extract produces a green colour with borax solution.

5. On addition of boric acid a reddish-brown colour is formed which, on addition of alkalies, changes to greenish-blue.

6. A piece of filter paper is impregnated with an alcohol extract, dried, and then moistened with boric acid solution slightly acidified with h ydrochloric acid, and redried. Pink or brownish-red colour is developed on the filter paper which becomes deep blue on addition of alkali.

Therapeutic uses: Turmeric is used as aromatic, antiinflammatory, stomachic, uretic, anodyne for billiary calculus, stimulant, tonic, car minative, blood purifier, antiperiodic, alterative, spice, colouring agent for ointments and a common household remedy for cold and cough. Externally, it is used in the form of a cream to improve complexion. Dye-stuff acts as a cholagogue causing the contraction of the gall bladder. It is also used in menstrual pains. Curcumin has choleretic and cholagogue action and is used in liver diseases. Curcumin is a nontoxic authorized colour, heat resistant and sensitive to changes in pH. Curcuminoids have antiphlogistic activity which is due to inhibition of leukotriene biosynthesis. ar-Turmerone has antisnake venom activity and blocks the haemorrhagic effect of venom.

Adulteration: The genuine drug is adulterated with the rhizomes of Acorus calamus.