Bharati Vidyapeeth University Biotechnology Practical Exam 2024-2025 PDF

BHARATI VIDYAPEETH (DEEMED TO BE UNIVERSITY) RAJIV GANDHI INSTITUTE OF INFORMATION TECHNOLOGY AND BIOTECHNOLOGY Katraj, Pune-411046 CERTIFICATE This is to certify that _____________________________________________...

BHARATI VIDYAPEETH (DEEMED TO BE UNIVERSITY) RAJIV GANDHI INSTITUTE OF INFORMATION TECHNOLOGY AND BIOTECHNOLOGY Katraj, Pune-411046 CERTIFICATE This is to certify that _________________________________________________ Roll no. _____ of M.Sc. Biotechnology SEM III has satisfactorily completed the required number of practical as laid down in the subject of Agricultural Biotechnology by the BHARATI VIDYAPEETH DEEMED TO BE UNIVERSITY for academic year 2024-2025. In-Charge External Examiner Principal 1  INDEX Sr. No. Experiment Page no. 1 Lab safety and study of equipments along with layout of tissue culture 03 lab. 2 Preparation of MS media along with the preparation of stocks of ‘PGR’. 09 3 Micropropagation of cardamom (Elettaria cardamomum) through in vitro 12 culture techniques. 4 Petal culture of Pomegranate (Punica granatum). 17 5 21 To perform cell suspension culture of hibiscus petals (Hibiscus rosa- sinensis). 6 Isolation and Culture of Anthers from Unopened Pomegranate Buds for 24 Haploid Plant Production. 7 Inoculation of immature zygotic lima beans (Phaseolus lunatus) for 27 embryo culture. 8 Regeneration of pelargonium (P. graveolens) through leaf culture 30 /somatic embryogenesis. 9 To induce callus using MS medium with 2,4-D acid on a carrot root 34 10 To performing the subculturing of spathyphylum plantlets. 37 2  EXPERIMENT - 1 AIM: Lab safety and study of equipments along with layout of tissue culture lab SAFETY GUIDELINES: 1. Wear lab coats to protect your clothing and minimize contamination risks. 2. Use disposable gloves when handling culture, chemical or during aseptic procedures. 3. Protect your eyes when working with chemicals especially during media preparation. 4. Wear masks when working in laminar flow hoods to prevent contamination from breathing. 5. Ensure all tools, culture vessels, and media are sterilized before use, typically using an autoclave. 6. Wash hands thoroughly before and after working in lab, especially after handling cultures or chemicals. 7. Dispose of biological waste, such as used cultures, in autoclave bag and sterilize before disposal. 8. Prohibit eating, drinking, or storing food in the lab to prevent contamination and chemical exposure. DO’s : 1. Always listen carefully to instructions given by lab technician and strictly follow all such guidelines. 2. Report any safety risks or incidents immediately to lab technician. 3. Always record your observation in worksheet provided in lab manual. 4. Always keep your mobile phones in the bags in switch off mode. 5. Return all the apparatuses or glass wares to the concerned lab technician after completion of experiments. 6. Do not throw tissue papers or cotton in the sink. Discard these properly. DON’Ts : 1. Never talk loudly or unnecessarily in the lab. 2. Do not attend mobile phones while working in the lab. 3. Do not eat or drink in the plant tissue culture lab. 4. Never take any culture or chemicals outside the lab. However, if there is a need to do so, take prior permission from the lab technician. 3  LAYOUT OF PLANT TISSUE CULTURE LAB 1. Media preparation room The media preparation room in a plant tissue culture lab is crucial for preparing and sterilizing culture media. The work surface of this room consist of preparation area which provides counter space for weighing chemicals, mixing media, and transferring solutions and also ensure surfaces are cleanable and resistant to spills. Other than preparation area there is stirring and mixing area which provide space for hot plates with magnetic stirrers or magnetic stirrer plates for mixing and dissolving media components. 2. Sterile handling area Sterile work should be located in a quiet part of the tissue culture laboratory and should be restricted to tissue culture and there should be not through traffic or other disturbances that is lightly to cause dust. The work area in its simplest form should be plastic laminate –topped bench preferably plain white or neutral grey to facilitate the observation of culture. 4 3. Culture/Growth room The culture room in plant tissue culture lab is designated to provide control environmental condition for the growth of plant culture. It maintains specific temperature, humidity and light condition optimal for plant growth. It is equipped with fluorescent or LED light for photosynthesis. Lighting conditions including intensity and photoperiod are control to mimic natural condition. It contains racks that hold culture vessels at various height to maximize space and light exposure. 4. Storage room The storage room in plant tissue culture lab is used for organizing and storing essential materials like chemical storage, glassware storage and cold storage. For chemical storage shelves or cabinets for storing media components, reagents and chemicals obtained with clear labeling. For glassware storing cabinets or shelves for clean glassware and equipment such as beaker, flask, and petri dishes. 5  5. Washing area The need for an extensive washup is minimize if disposable plastics are used but there will be need for some glassware washing. Washup and sterilization facilities are best situated outside the tissue culture lab as the humidity and heat that they produce nay be difficult to dissipate. EQUIPMENTS : 1. Laminar air flow Laminar air flow (LAF) hood in PTC lab provides a sterile working environment by filtering and directing air through a HEPA (High Efficiency Particulate Air) filter. This prevents contamination of plant tissues and culture media by removing air borne particles and microorganisms. The LAF hood ensure that any procedures involve in open culture vessels or sensitive plant material are conducted in clean control environment, reducing the risk of contamination and increasing the success of tissue culture experiment. 2. Autoclave An autoclave is a crucial piece of equipment used for sterilization. It operates on the principle of using high-pressure steam to kill microorganisms, including bacteria, fungi, viruses, and spores, which might be present on culture media, tools, and other equipment. This ensures that all materials are free from contaminants before they come into contact with plant tissues. The autoclave generates steam by heating water to a high temperature. The steam is then introduced into the chamber where the items to be sterilized are placed. The autoclave operates at a pressure of approximately 15 psi (pounds per square inch) and a temperature of 121°C (250°F). This combination effectively kills most microorganisms within a specified time, typically around 15-30 6  minutes, depending on the load and type of material. It ensures that culture media are free from microbial contamination, which is critical for successful plant growth and development. 3. Distillation unit A distillation plant in a tissue culture lab is used to produce high- purity water, which is essential for preparing culture media and other solutions. Pure water is critical to avoid contamination and ensure consistent results in tissue culture experiments. 4. pH meter A pH meter in a plant tissue culture (PTC) laboratory is an essential tool used to measure and adjust the pH of culture media. Accurate pH is crucial because it affects nutrient availability, enzyme activity, and overall plant growth. Before autoclaving, the pH of the media is typically adjusted to an optimal range (usually between 5.6 and 5.8) using a pH meter. This ensures a conducive environment for tissue cultures, promoting successful plant development. Regular calibration of the pH meter is necessary to maintain its accuracy and reliability. 5. Weighing Balance A weighing balance in a plant tissue culture (PTC) laboratory is used to precisely measure the amounts of chemicals, growth regulators, and other media components required for preparing culture media. Accurate weighing is critical to ensure the correct concentration of ingredients, which directly influences the success of tissue cultures. Balances in a PTC lab are typically highly sensitive, capable of measuring small quantities with precision, often down to milligrams or micrograms, ensuring consistency and reproducibility in experiments. 6. Refrigerator A refrigerator in a plant tissue culture (PTC) laboratory is used to store temperature- sensitive materials such as prepared culture media, plant growth regulators, chemicals, and some plant samples. By keeping these items at low temperatures (usually around 4°C), the refrigerator helps preserve their stability and effectiveness, preventing degradation or spoilage. This ensures that materials remain viable for use in tissue culture procedures, contributing to consistent and successful outcomes. 7 7. Microscope A microscope in a plant tissue culture (PTC) laboratory is essential for observing and analyzing plant tissues at a cellular level. It is used to examine the health, structure, and development of cultured tissues, as well as to detect any signs of contamination or abnormalities. Microscopes enable researchers to monitor cell division, differentiation, and other critical processes, providing valuable insights into the success of tissue culture experiments and guiding adjustments to protocols when necessary. Simple, compound, inverted binocular dissection microscopes are essential for various purposes. 8. Hot air oven A hot air oven in a plant tissue culture (PTC) laboratory is used for the dry sterilization of glassware, metal instruments, and other heat-resistant materials. By exposing items to high temperatures (typically 160-180°C) for a specified period, the oven effectively destroys microorganisms, ensuring that tools and equipment are sterile and ready for use in aseptic procedures. Additionally, hot air ovens can be used for drying materials after washing or for dehydrating samples. Their reliable sterilization process is crucial for maintaining a contamination-free environment in the lab. 8  EXPERIMENT - 2 AIM: Preparation of MS media along with the preparation of stocks of ‘PGR’. THEORY: Media is an artificial system that is provided to the plants parts for nutrition in plant tissue culture. Formulated media powders are used in appropriate amount to make a desired media for plant tissue culture. Preparation of MS media involves preparation of stocks for macronutrients, micronutrients, Ion source and vitamin source. Relatively large amounts of some inorganic elements (the so-called major plant nutrients: - ions of nitrogen (N), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg) and sulphur (S); and small quantities of other elements (minor plant nutrients or trace elements: - iron (Fe), nickel (Ni), chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo). These elements with carbon (C), oxygen (O) and hydrogen (H) makes the 17 essential elements. Certain others, such as cobalt (Co), aluminium (Al), sodium (Na) and iodine (I), are essential or beneficial for some species. The most commonly used medium is the formulation of Murashige and Skoog (MS) (1962). This medium was developed for optimal growth of tobacco callus and the development involved a large number of dose- response curves for the various essential minerals. Each plant species has its own characteristic elementary composition which can be used to adapt the medium formulation. These media result often in a much-improved growth. A major problem in changing the mineral composition of a medium is precipitation, which may often occur only after autoclaving because of the endothermic nature of the process. Plant tissue culture media provides these inorganic nutrients and usually a carbohydrate (sucrose is most common) to replace the carbon which the plant normally fixes from the atmosphere by photosynthesis. To improve growth, many media also include trace amounts of certain organic compounds, notably vitamins, and plant growth regulators. Plant tissue culture media are therefore made up from solutions of the following components: macronutrients (always employed). micronutrients (nearly always employed but occasionally just one element, iron, has been used. plant growth regulators (nearly always added). vitamins (generally incorporated, although the actual number of compounds added, varies greatly). sugar (nearly always added, but omitted for some specialised purposes). a solidifying agent (used when a semi-solid medium is required. Agar or a gellan gum are the most common choices). amino acids and other nitrogen supplements (usually omitted, but sometimes used with advantage). buffers (have seldom been used, but the addition of organic acids or buffers could be beneficial in some circumstances). Inorganic nutrients are added to plant culture media as salts. In weak aqueous solutions, such as plant media, salts dissociate into cations and anions. Thus calcium, magnesium and potassium are absorbed by plant cells (normally those of the root) as the respective cations Ca2+, Mg2+ and K+, nitrogen is mainly absorbed in the form nitrate (the anion, NO3 - ) 9 although uptake of ammonium (the cation, NH4 + ) may also occur, phosphorus as the phosphate ions HPO4 2- and H2PO4 - ; and sulphur as the sulphate ion SO4 2-. In tissue culture, uptake is generally proportional to the medium concentration up to a concentration of twice MS. TABLE FOR THE COMPOSITION OF THE MS MEDIA: Concentration in Volume of stock Concentration in Component Stock per litre of medium (mg/L) (mg/L) medium (L) Macronutrients 20x 1x NH4NO3 33000 1650 KNO3 38000 1900 CaCl2.2H2O 8800 440 50 MgSO4.7H2O 7400 370 KH2PO4 3400 170 Micronutrients 200x 1x KI 166 0.83 H3BO3 1240 6.2 MnSO4.4H2O 4460 22.3 ZnSO4.7H2O 1720 8.6 5 Na2MoO4.2H2O 50 0.25 CuSO4.5H2O 5 0.025 CoCl2.6H2O 5 0.025 Iron Source 200x 1x FeSO4.7H2O 5560 27.8 5 Na2EDTA.2H2O 7460 37.3 Vitamins 200x 1x Myo-inositol Add freshly 100 Nicotinic acid 100 0.5 Pyridoxine-HCL 100 0.5 5 Thiamine-HCL 100 0.5 Glycine 400 2 Carbon Source (3%) Add freshly to the Sucrose Adjust pH to 5.7-5.8 before autoclaving medium 30g/L Solidifying Agent Agar Agar Add freshly to the medium 14g/L Macronutrients- Includes six major components: N, P, K, Ca, Mg, S Nitrates and ammonia needed. Micronutrients- Requires in small amount: Fe, Mn, B, Cu, Mo Iron and Zinc are commonly used in preparing culture media. Co, I, Na requires for proper growth 10 Carbon Source- Sucrose- Good growth. Enhance proliferation of cells. Regeneration of shoots. Vitamins- Thiamine is an essential for many plant cells. Other mentioned vitamins stimulate the growth of cells in some cases. PROCEDURE: 1. Stocks of Macronutrients, micronutrient, vitamins and Iron source were prepared. Fig: Stocks A, B, C and D respectively a) NH4NO3, KNO3, CaCl2.2H2O, MgSO4.7H2O and KH2PO4 were measured in appropriate amount for the preparation of macronutrients stock(20x). b) KI, H3BO3, MnSO4.4H2O, ZnSO4.7H2O, Na2MoO4.2H2O, CuSO4.5H2O and CoCl2.6H2O were measured in appropriate amount for the micronutrients stock (200x). c) FeSO4.7H2O and Na2EDTA.2H2O were measured in appropriate amount for the preparation of Iron source stock (200x). d) Mayo-inositol, Nicotinic acid, Pyridoxine HCl, Thiamine HCl and Glycine were measured in appropriate amount for the preparation of Vitamin stock (200x). e) 3% Sucrose was used in the freshly prepared media. 2. These stocks were used to prepare MS media freshly using the standard table. 3. Autoclaved was kept for pre-heating. 4. Stocks were added to the freshly prepared sucrose solution. 5. Mg of Agar powder was added for solidification. 6. Media was cooled and filled in media bottles. 7. Tissue paper, cotton balls, steel petri plates, forceps, distilled water and media was autoclaved at 121° C and 15 psi for 20 mins. 11  EXPERIMENT - 3 AIM: Micropropagation of cardamom (Elettaria cardamomum) through in vitro culture techniques. THEORY: Micropropagation is one of the most popular techniques of tissue culture. It is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods. Micropropagation is used to multiply novel plants, such as those that have been genetically modified or breed through conventional plant breeding methods. It is also used to provide a sufficient number of plantlets for planting from a stock plant which does not produce seeds, or does not respond well to vegetative reproduction. Cardamom (Elettaria cardamomum) is a tall perennial shrub of the family Zingiberaceae. The aerial part of the plant consists of 10 to 20 leafy shoots (pseudostems) that may attain a height up to ~5 m. The plant is valued for its dried fruits. The aromatic seeds are used as a popular spice and flavouring agent, besides possessing carminative, stomachic, and antimicrobial properties. For cardamom, the principle of micropropagation involves several key steps and concepts. Totipotency refers to the potential of a single plant cell to regenerate into a complete, genetically identical plant. This is achieved through a sequence of cellular dedifferentiation and redifferentiation. In micropropagation, this principle is harnessed by isolating and culturing plant tissues under controlled conditions. Plant tissue cultures are grown on a nutrient medium that provides essential nutrients, vitamins, and growth regulators (hormones). The commonly used medium for cardamom micropropagation is the Murashige and Skoog (MS) basal medium. This medium supports the growth and development of plant tissues by supplying macronutrients (e.g., nitrogen, phosphorus, potassium), micronutrients (e.g., iron, manganese, zinc), vitamins (e.g., thiamine, nicotinic acid), carbohydrates (e.g., sucrose), and growth regulators. Plant growth regulators are critical in controlling the differentiation and development of tissues. The main types of growth regulators used in cardamom micropropagation are: Cytokinin (e.g., Benzylaminopurine, BAP and Kinetin). The process of micropropagation of cardamom involves several distinct stages such as selection of suitable explants such as plant part including the meristem region from healthy cardamom plants and surface sterilization to eliminate surface contaminants using disinfectants like ethanol and sodium hypochlorite. Inoculation of the sterilized explants was done onto an MS Basal medium supplemented with BAP and Kinetin to induce root and shoot formation. REQUIREMENTS: Plant Material: Cardamom explant (meristem containing portion shoot base) Sterilizing Agents: 2% Fungicide, 2% Cetrimide, 0.5% Sodium Hypochlorite, 70% Ethanol Culture Media: Murashige and Skoog (MS) basal medium, supplemented with growth regulators such as Benzylaminopurine (BAP) and Kinetin Equipment: Laminar airflow hood, autoclave, forceps, scalpel, sterile double distilled water 12 PREPARATION OF PGR (1 mg/mL): Dissolve 10 mg BAP and Kinetin in 0.5 mL ethanol. Add 9.5 mL double D/W and make up the volume to 10 ml. PREPARATION OF SURFACE STERILIZATION CHEMICALS: 1. 2% Fungicide (1L) - Weigh 20 gm fungicide powder and dissolve in 1000 mL double D/W 2. 2% Cetrimide (1L) - Weigh 20 gm cetrimide powder and dissolve in 1000 mL double D/W. 3. 0.5% Sodium hypochlorite (1L) - Weigh 40 gm NaOCl2 and dissolve in 1000 mL double D/W. 4. 70% Ethanol - Measure 70 mL of 100% ethanol using a measuring cylinder. Transfer the measured ethanol into beaker. Add 30 mL of distilled water to the beaker containing ethanol. Fig: Surface sterilization chemical bottles (2)% Fungicide, 2% Cetrimide, 0.5% NaOCl2 PROCEDURE: Select cardamom explant. Excise plant part including the meristematic region. Wash the explant thoroughly under running tap water for 5 minutes. Wash with sterile distilled water twice. Surface sterilize the explant by immersing them in 2% fungicide with continuous shaking for about 20 minutes. Wash with sterile distilled water twice. Surface sterilize again with 2% cetrimide with continuous shaking for 10 minutes. Wash with sterile distilled water twice. Keep the explant in 70% ethanol for 30 sec. Transfer the explants to a solution of 0.5% sodium hypochlorite for 8 minutes with continuous shaking. Rinse the explants 3 times with sterile distilled water to remove any traces of sterilizing agents. Clean the LAF with 70% alcohol and sterile cotton. 13  Clean the petri plates and keep the instruments in 100% alcohol. Flame before use. Cut the explant at the extreme ends to remove the damaged cells. Clean the blackened portion of the explant as much as possible to remove dead cells. Place it in prepared media bottle with the shoot part facing upwards. Seal the media bottle and incubate them in a growth room at 25℃. OBSERVATION: Fig 1: Mother plant cardamom Fig 2: Meristematic region of the plant (Elettaria cardamomum) Fig 3: Explant including meristem Fig 4: Inoculation of explant in media containing shoot base bottles 14 Fig 5: Rooting and shooting on 14th day Fig 6: Growth on 28th day OBSERVATION TABLE I: Control Shoot/ Contamination Dead Browning Growth Bottle No Root 21 ✔ × × × ✔ 22 ✔ ✔ × × ✔ 39 ✔ ✔ × × ✔ 28 × ✔ × ✔ × 31 × ✔ × ✔ × 35 × × × ✔ × PGR Shoot/ Contamination Dead Browning Growth (BAP+Kinetin) Root Bottle No 37 × ✔ × ✔ × 32 ✔ ✔ × × ✔ 29 ✔ ✔ × × ✔ 15  27 ✔ × × × ✔ 24 ✔ ✔ × × ✔ 25 ✔ ✔ × × ✔ 38 ✔ ✔ × × × INDEX: ✔= Presence ×= Absence 16  EXPERIMENT - 4 AIM: Petal culture of Pomegranate (Punica granatum). THEORY: The pomegranate (Punica granatum L.) is often recognized under the Punicaceae family, which consists of two species Punica granatum and P. protopunica and a single genus, Punica. Because of its delicious fruits, medicinal and ornamental uses, and global distribution, it is a species of great commercial importance. Fruit trees that are micropropagated can produce true-to-type plants, overcome the challenges of vegetative propagation, and produce planting materials quickly and in large quantities. Pomegranate plants can be propagated via culture, which is advantageous if we want to make sure the offspring are disease-free and genetically identical to the parent plant. An essential component of pomegranate (Punica granatum) research and growth was cultivation. The term "in vitro culture" describes the cultivation of plant tissues, cells or organs under highly controlled conditions, usually in a lab. In situations when more conventional techniques, such as using seeds or cuttings, may not be appropriate, this method is useful for growing uniform, disease-free plants. The micropropagation of pomegranate is practiced by using explants such as shoot tips, nodal segments, axillary buds , etc. The use of petals for micropropagation is an unusual practice as petals are part of flower that are not involved in process of plant regeneration because they do not contain the type of cells that are capable of differentiating into whole plant. In this experiment capacity of callus induction by petals of pomegranate flower buds is assessed. As the flower is not fully developed it may have capacity to regenerate and form callus. Callus is dedifferentiate mass of cells that has capacity to develop into whole plant. Murashige and Skoog (MS) media were used for the callus induction. The MS media was supplemented with 2,4- Dichlorophenoxyacetic acid(2,4-D) which is a synthetic auxin. Auxins are plant hormones that regulate cell growth and division. Using 2,4-D as supplement may induce the cells in petals to dedifferentiate and form callus which can be then used for further propagation. REQUIREMENTS: Plant material: Unopened flower buds of pomegranate plant. Sterilizing agents: 2% Fungicide, 2% Cetrimide, 0.5% Sodium Hypochlorite, 70% Ethanol Culture Medium: Murashige and Skoog (MS) basal medium, supplemented with 2,4- Dichlorophenoxyacetic acid(2,4-D) which is a synthetic auxin Equipment: Laminar airflow hood, autoclave, forceps, scalpel, sterile double distilled water, PTC bottles. 17 Preparation of Sterilizing agents i. 2% Fungicide: 4gm of SAAF/Bavistin in 200ml of distilled water. ii. 2% Cetrimide: 4gm of cetrimide powder in 200ml of distilled water. iii. 0.5% sodium hypochlorite: 0.5gm of NaOCl₂ in 100ml distilled water. iv. 70% Alcohol: 70ml alcohol in 30 ml of distilled water. Preparation of Culture medium For preparation of 1L of culture medium mix 65ml MS basal salts, 30gm sucrose and 14gm agar powder (usually 8gm/L is used but as agar’s quality is decreased 14gm/L has been used) with 935ml of distilled water. Dissolve the mixture by heating it. After heating add 2mg/L 2,4-D. Pour the medium into PTC bottles and autoclave medium at 121℃ for 20 min at 15 psi. PROCEDURE: Sterilization of plant material Take unopened pomegranate flower buds into a PTC bottle and wash them with clean water. Add 2% fungicide to the bottle and treat it for 10 min while shaking. After 10 min wash the buds with double distilled water. Add 2% cetrimide for 5 min while shaking. Wash buds with double distilled water after 5 min. Add 0.5% sodium hypochlorite for 1 min and wash the buds with double distilled water. Inoculation Prepare the laminar air flow cabinet (LAF) by cleaning it with 70% alcohol and arrange all required materials in the cabinet and turn on the UV for 45 min. Turn off the UV after 45 min and treat the buds with 70% alcohol for only 30 seconds. Take the flower bud on a steel plate and cut open the flower bud. Dissect the petals and separate them from anthers. Inoculate the petals into the culture medium such that they only slightly touch the medium. Label the PTC bottle including all necessary details like name of explant, date of inoculation and culture medium used. Incubate the Culture into a incubation room at 25℃. Observe the culture at different time intervals. 18 PLANT PARTS USED AND PROCEDURE: Fig 1: Unopened pomegranate flower buds Fig 2: Flower buds kept in D/W after washing Fig 3: Dissection of petals from flower bud Fig 4: Flower bud ready for dissection after sterilization 19 OBSERVATIONS: Fig 5: Inoculation of petals into Culture medium Fig 6: Petals after 72 hrs of incubation The unopened flower buds when dissected contained immature anthers that were further used for anther culture. The petals were not developed fully and were reddish orange in colour when dissected. After incubation for about 72 hrs the petals looked dry and didn’t show any outgrowth. The colour of petals had changed from reddish orange to slightly brown indicating death of the tissue and cells. There were no signs of contamination in the culture bottle although water droplets were present on the side of the bottle. 20  EXPERIMENT - 5 AIM: To perform cell suspension culture of hibiscus petals (Hibiscus rosa-sinensis). PRINCIPLE: The Hibiscus plant commonly known as China rose and, scientifically known as Hibiscus rosa-sinensis, is a flowering plant native to tropical and subtropical regions. It is known for its large, showy flowers that come in various colours such as red, pink, yellow, and orange. Here are some key aspects of the Hibiscus plant and its medicinal uses: Hibiscus tea, made from dried hibiscus flowers, is consumed in many cultures for its digestive properties. Hibiscus is known for its potential to lower blood pressure. Hibiscus contains antioxidants such as flavonoids, polyphenols, and anthocyanins. These compounds help combat oxidative stress in the body. Hibiscus extracts are used in cosmetics and skincare products for their moisturizing, anti-aging, and exfoliating properties. They can help improve skin texture and promote a youthful appearance. Callus cell suspension is a technique used in plant tissue culture where a callus, an unorganized plant cell mass, is maintained and propagated in a liquid medium. The process involves transferring the callus to a fluid culture medium, allowing the cells to grow and multiply in suspension rather than on a solid medium. Liquid cultures are constantly agitated, generally by a gyratory shaker at 100-250 ppm. This enhances the aeration and dissociation of cell clumps into smaller pieces. This technique is beneficial for large-scale production of plant cells and secondary metabolites. REQUIREMENTS: Chemicals: MS media, Picloram, 2,4-D Glassware: 100ml conical flask Instruments: Rotary shaker, Laminar Air Flow, Autoclave Miscellaneous: Forceps PROCEDURE: 1. Media preparation: Prepare 500ml of media containing MS + Picloram(2mg/l) + 2,4-D(1mg/l). 2. Transfer a small lump of friable callus into a 100 ml conical flask containing 50 ml of prepared suspension media. 3. It is agitated on a rotary shaker at 120 rpm until it grows. 4. Observe the suspension culture every day and record your results. 21  Fig 1: Callus of hibiscus Fig 2: Callus in suspension media OBSERVATION TABLE I: Bottle no: Compacted callus Non-compacted callus 22   24   28   29   30   32   33   35   36   37   22 OBSERVATIONS: Fig.3: Non-compact callus Fig.4: Compact callus 23  EXPERIMENT - 6 AIM: Isolation and Culture of Anthers from Unopened Pomegranate Buds for Haploid Plant Production. THEORY: Anther culture is a type of tissue culture technique used to produce haploids and diploids. It is similar than the pollen culture technique. It uses microspores or anthers for plant regeneration. During the process, anthers are excised at a critical stage from an unopened flower bud aseptically. Then they are cultured on a nutrient medium for the formation of callus tissue or embryoids that gives rise to haploid plantlet through embryogenesis or organogenesis. This is a technique by which immature pollen is made to divide and grow into tissue (either callus or embryonic tissue), primarily to produce haploids (plants with an N chromosome number) known to be anther culture. Haploid production through anther culture has been referred to as androgenesis. Culturing through anthers or microspores is one of the most popular methods for the production of haploids on artificial culture medium. There are several methods for haploids production but their occurrences are very rare. In this process pollen-containing anthers are isolated from a flower and put into a suitable culture medium. During this process some microspheres survive and develop into tissue. Some yield into embryonic tissue, which when supplemented with favorable medium, is further utilized for shoot and root development. But if callus appears, after hormonal treatment it will differentiate into shoot and root tissue. REQUIREMENTS: Chemicals: 2% fungicide 2% cetrimide 70% alcohol MS (Murashige and skoog) media Sterile distilled water Materials: Pomegranate buds Plant tissue culture bottles Forceps, Blade, Petri dish Cotton, stirrer beaker Media required: MS Basal + 2,4-D(2mg/lit) – 1 L PROCEDURE: Take the pomegranate flower and wash under the tap water twice. Give the treatment of 2% fungicide for 10 min with continuous shaking. 24  discard the fungicide and wash with double distilled water twice. Give the treatment of 2% cetrimide, shake for 5 min and wash with double distilled water twice. Give the treatment of 70% alcohol inside the laminar air flow cabinet for 30 sec. Wash with double distilled water once. In laminar air flow cabinet, peel the flower with the help of Blade. Remove the outer layer Separate the anther from the flower and inoculate onto the MS media. OBSERVATIONS: Fig1: Pomegranate buds Fig2: Separation of anther from bud 25  (a) (b) (c) Fig3: Inoculated anthers (a), (b) and (c) 26  EXPERIMENT - 7 AIM: Inoculation of immature zygotic lima beans (Phaseolus lunatus) for embryo culture. THEORY: Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro, with the goal of obtaining a viable plant. There are two types of embryo culture, (a) mature embryo culture and (b) immature embryo culture. The mature embryo culture is the culture of mature embryos derived from ripe seeds. This type of culture is done when embryos do not survive in vivo or become dormant for long periods of time. Seed dormancy of many species is due to chemical inhibitors or mechanical resistance present in the structures covering the embryo, rather than dormancy of the embryonic tissue. Excision of embryos from the testa and culturing them in the nutrient media may bypass such seed dormancy. In the immature embryo culture, the immature embryos are rescued from wide crosses. Wide hybridization, where individuals from two different species of the same genus or different genera are crossed, often leads to failure. Hence, immature embryo culture is mainly used to avoid embryo abortion with the purpose of producing a viable plant. The morphology and anatomy of Lima bean (Phaseolus lunatus) seeds are notable for their structure and organization, which play important roles in their germination, development, and adaptation to various environmental conditions. Lima bean seeds are generally kidney-shaped or somewhat flattened. They vary in size depending on the cultivar and maturity but typically measure around 1.5 to 2.5 centimetres in length. Application of embryo culture includes the following: 1. Prevention of embryo abortion in wide crosses 2. Production of haploids 3. Overcoming seed dormancy 4. Shortening of breeding cycle 5. Prevention of embryo abortion with early ripening stone fruits 6. For in vitro clonal propagation REQUIREMENTS: Plant Material: Lima Seeds Chemicals and Reagents: 2% Fungicide, 2% Cetrimide, 0.5% Sodium Hypochlorite, 70% Alcohol, 100% Alcohol Culture Media: Murashige and Skoog (MS) basal medium Equipment: Laminar airflow hood, autoclave, forceps, scalpel, Petri dish Miscellaneous: Cotton, Distilled Water, Double distilled water, Beaker, Pipettes, Culture bottles PROCEDURE: 1. Remove seeds from lima pods. 2. Wash seeds under running tap water. 27  3. Wash with 2% fungicide, shake for 5 minutes, and rinse with double-distilled water twice. 4. Wash with 2% cetrimide, shake for 5 minutes, and rinse with double-distilled water twice. 5. Wash with 0.5% sodium hypochlorite, shake for 2 minutes, and rinse with double- distilled water twice. 6. Wash with 70% alcohol for 5 seconds in a laminar air flow cabinet and rinse with double-distilled water once. 7. Inoculate seeds onto Murashige and Skoog (MS) media aseptically. 8. Seal the media bottle and incubate the cultures at 25℃. OBSERVATIONS: Fig 1: Lima seeds Fig 2: Germination on 2nd day Fig 3: Growth on 9th day 28 OBSERVATION TABLE I: Bottle No Shoot/ Root Contamination Growth 21 ✔ ✔ ✔ 22 × ✔ ✔ 24 × ✔ × 25 × ✔ × (dark room) 27 ✔ × ✔ 28 × ✔ × 29 × × ✔ (dark room) 30 ✔ ✔ ✔ 31 × ✔ × 32 × ✔ × 33 ✔ ✔ ✔ (dark room) 34 × ✔ × (dark room) 35 × ✔ × 36 × ✔ × 37 × ✔ × 38 × ✔ × 39 × ✔ × ✔= Presence × = Absence 29  EXPERIMENT - 8 AIM: Regeneration of pelargonium (P. graveolens) through leaf culture /somatic embryogenesis. THEORY: Pelargoniums are often called geranium. They fall within the plant family Geraniaceae. Pelargonium is native to South Africa; pelargonium species were introduced to Europe in the 17th century. They quickly become popular in gardens due to their aromatic foliage and attractive flowers. Pelargonium graveolens is a erect, multi-branched shrub. The leaves are deeply incised, velvety, and soft to touch. The flowers vary from pale pink to white. It has great importance in the perfume industry. Pelargonium distillates and absolutes commonly known as geranium oil and its used for aromatherapy and massage therapy application. Flowers and leaf are used in cakes, jams, jellies, ice-creams, salad, tea and sugars. They are often used in traditional medicine. The Geranium oil possesses antimicrobial, antioxidant, pesticidal properties. They were used as insects repellent, strong fragrance is effective in repelling the mosquitos and some insects. Somatic embryogenesis is a process in which somatic cells are used to developing a embryo that can eventually grow into full plants. This technique widely used in plant tissue culture for the propagation and genetic improvement of plant. Somatic embryogenesis in pelargonium involves including leaf to undergo a series of developmental changes leading to formation of embryo that can be cultured to produce a new plant. REQUIREMENTS: Plant material: Scanted geranium (pelargonium) Surface sterilizing chemicals: 2% fungicide, 2% cetrimide, 0.5% sodium hypochloride, 70% ethanol and sterile D/W. Other: Laminar air flow, steel plates, forceps, blades, cotton, tissue paper, bottles etc. PREPARATION OF SURFACE STERILISATION CHEMICALS: I) 2% Fungicide (1L): Weigh 20 gm fungicide powder and dissolve in 1000mL double D/W II) 2% Cetrimide (1L): Weigh 20 gm cetrimide powder and dissolve in 1000mL double D/W. III) 0.5% Sodium hypochlorite (1L): Weigh 40 gm NaOCl2 and dissolve in 1000mL double D/W. IV) 70% Ethanol: Measure 70 mL of 100% ethanol using a measuring cylinder. Transfer the measured ethanol into beaker. Add 30 mL of distilled water to the beaker. PROCEDURE: i) Select leaf of Pelargonium and keep it in the beaker containing water. 30  ii) Clean or wipe the LAF with 70% ethanol and keep media bottles, still plates, forceps, blade, cotton and tissue paper bottles and alcohol bottles in LAF. After turn on the U.V. light for 45 mins. A) Process of surface sterilization: i) Wash the explant thoroughly under running tap water for 5 minutes. ii) Wash with sterile distilled water twice. iii) Surface sterilize the explant by immersing them in 2% fungicide with continuous shaking for about 15 minutes. iv) Wash with sterile distilled water twice. v) Again give the treatment of 2% cetrimide with continuous shaking for 5 minutes. vi) Wash with sterile distilled water twice. vii) Transfer the explants to a solution of 0.5% sodium hypochlorite for 3 minutes with continuous shaking. viii) Wash with the sterile D/W. ix) Keep the explant in 70% ethanol for 5sec (in LAF). x) Rinse the explant 2 times with sterile distilled water to remove any traces of sterilizing agents. B) Process of Inoculation: i) Clean the plates with tissue paper. ii) Take explant on tissue paper and dry the explant. iii) Remove the tissue paper and cut the extra part of leaf, take only midrib portion of leaf (remove petiole and extra part of leaf except midrib). iv) Cut the leaf into small pieces and inoculate the explant onto the media. v) Keep it in the growth room. PLANT PARTS Fig 2: Explant Fig1: Mother plant 31 OBSERVATION: Fig 3: Inoculation of explant in media bottle Fig 4: Yellowing of leaves on the 9th day OBSERVATION TABLE: Control bottle no Contamination Yellowing at Dead surface Control 21 ✔ × ✔ 37 ✔ × ✔ Ms+ NAA × ✔ 22 ✔ × ✔ 24 ✔ × ✔ 25 ✔ × ✔ 27 ✔ × ✔ 28 ✔ × ✔ 29 ✔ × ✔ 30 ✔ × ✔ 31 ✔ × ✔ 32 ✔ × ✔ 33 ✔ × ✔ 34 ✔ × ✔ 35 ✔ × ✔ 36 ✔ × ✔ 37 ✔ × ✔ 38 × ✔ 39 ✔ × ✔ 32 INDEX: × = absence ✔ = presence 33  EXPERIMENT - 9 AIM: To induce callus using MS medium with 2,4-D acid on a carrot root THEORY: The carrot (Daucus carota) is a member of Umbelliferae, a biennial plant grown with an edible taproot known for its medicinal uses. Carrots are a source of vitamin A and fiber for human nutrition. Carrot is a model plant as it is a reliable material for in-vitro and somatic embryo cultures. Synthetic auxin 2,4-Dichlorophenoxy acetic acid(2,4-D) can be used to establish embryogenic callus and for suspension culture in carrots. The segments of various plant organs from many species of plants inoculated onto sufficient media form relatively undifferentiated tissue (callus) containing primarily clusters of parenchymatous cells. Rapid cell divisions appear especially on the segments consisting of the cells of the primary or secondary meristem (shoot or root apical meristem, cambium, felogen). Differentiated mature cells produce callus to a lesser extent. Carrot production and regeneration are affected by genotype, type of explants, type, and concentration of hormones applied in the medium. Auxins are widely used to induce callus production. The most frequently used auxin to initiate carrot cultures is 2,4-D. For breeding purposes to produce embryogenic callus. The somatic embryogenesis process is often initiated in media containing high levels of auxins, especially 2,4-D. The explant was surface sterilized and inoculated on MS media supplemented with 2,4-D. REQUIREMENTS: Samples: Carrot root Chemicals: MS media, 2,4-D acetic acid, 2% SAAF, 2% Cetrimide, 70% ethanol, 0.5% sodium hypochlorite Glassware: Glass jar bottles, glass rods, glass beakers Instruments: Laminar Air Flow, Autoclave Miscellaneous: Scalpel blade, forceps, plate, cotton, tissue, sterile double distilled water PROCEDURE: 1. Select typically shaped, clean, healthy carrots. 2. Wash the roots under running tap water and clean them using a scalpel blade. 3. Cut the carrot into medium-sized pieces. 4. Surface sterilization of the explant has to be carried out, for this: Suspend the explant in 2% fungicide (SAAF) for 20 min with continuous shaking, later wash with sterile distilled water. Suspend in 2% Cetrimide for 10 min with continuous shaking, wash with sterile distilled water. Dip it in 70% ethanol for 30 sec Fig 1: Sterile carrot 34  Finally, suspend in sodium hypochlorite (0.5%) for 8 min with continuous shaking. 5. Rinse 3x for 3 minutes in sterile distilled water inside Laminar airflow. 6. Decant the water and transfer the segments into a sterile petri dish in a flow hood. 7. Cut 1 cm of the exposed surface of the segment off and discard. 8. Inoculate the piece into the MS medium with 2,4-D acetic acid. 9. Wrap the flask with parafilm and store it under sufficient exposure to light and temperature in the growth room. 10. Observe the culture daily and record the growth. PROCEDURAL STEPS: Fig 2: Carrot cut into explant Fig:3 Explant being transferred to media Fig 4: Explant on the surface of culture media 35 OBSERVATION Fig 2: Growth on carrot callus on 3rd day OBSERVATION TABLE I: Bottle No: Contamination Dead Browning Callus growth 22     28     25     27     30     31     33     35     37     36  EXPERIMENT - 10 AIM: To performing the subculturing of spathyphylum plantlets. THEORY: Subculturing is the process of removing plant cells from their older media and transferring them to a fresh medium. This enables further proliferation of the cells. This is mainly done when plants have completely outgrown their previous culture jar or the media is exhausted (or both factors). In such cases, plant tissues are divided into small sections are transferred to fresh media. The number of times the subculturing process is done, it’s known as the “propagation cycle”. Subculturing is done either at the initiation phase, where callus is transferred to a fresh media, and/or multiplication stage where shoots are divided and transferred to a fresh media. The process helps in the preservation of the dedifferentiated mass indefinitely. You must know that the callus obtained from the original or initial explant is known as the primary callus. The primary callus is further dissected in the process and transferred to a fresh media, generating a secondary callus, which is generally used for the preservation purposes of tissues for a longer time. The process of successive dissection and transfer of callus to a fresh media is known as subculturing and the time between initiation and transfer is known as passage. REQUIREMENTS: Plant Materials: spathyphylum Media: MS Basal + NAA (2mg/L) MS Basal + NAA (0.5mg/L) + BAP (4mg/L) Glasswares and equipments 1) Laminar air flow 2) forceps/blade/scalpel 3)Sterile dish 4) Sterile cotton swab and tissue paper 5) Alcohol - 70% & 100% 6) Sterile distilled water PROCEDURE: 1. Clean the LAF with 70% alcohol and sterile cotton 2. Clean the petri plates and keep the instruments in100% alcohol. 3. Flame the before use elect the culturing plant (spathyphylum) 4. Take the culturing plant in petri dish 5. Remove the unwanted part of culturing plant (spatiphylum) with the help of blade 6. Select the healthy plantlets in inoculates the fresh medium with the help of forceps 37 7. Seal the media bottle and incubate them in a growth room at 250C OBSERVATION: Fig 1: Mother plant (spathyphylum) Fig 2: Sub-cultured plant Fig3: Growth of spathyphylum OBSERVATION TABLE I: Control Contamination Dead Browning No Growth Bottle No 39 ✔ × × × 38 BAP + KN Bottle No Contamination/ Dead Browning No growth 32 ✔ × × 21 ✔ × × 2,4-D Bottle No No Dead Browning Contamination/ growth 22 ✔ - - 24 ✔ - - 25 ✔ - - 27 ✔ - - 29 ✔ - - 30 ✔ - - 31 ✔ - - 33 ✔ - ✔ 34 ✔ - - 36 ✔ - - 37 ✔ - - 38 ✔ - - 39