Plant Tissue Culture Lecture 1 PDF

Plant Tissue Culture (SBS4063) By Dr Nazariyah Yahaya Ph.D in Plant Science (Uni. of Sheffield, UK), MSc in Molecular Biology (UKM) BSc in Genetics (UKM)  Introduction and Historical Development of Cell and...

Plant Tissue Culture (SBS4063) By Dr Nazariyah Yahaya Ph.D in Plant Science (Uni. of Sheffield, UK), MSc in Molecular Biology (UKM) BSc in Genetics (UKM)  Introduction and Historical Development of Cell and Tissue Culture Techniques Definition of Plant Tissue Culture Plant tissue culture is a highly technical method of asexual plant propagation. It involves growing plantlets from small pieces of plant tissue taken from a parent plant. This process uses an artificial, sterile medium to cultivate the plant cells in a laboratory setting. The remarkable ability of a single plant cell to autonomously produce an entire new plant is known as totipotency. Additionally, the plasticity of plants allows them to adapt and thrive despite changes in their environment. Historical Developments of Cell and Tissue Culture Techniques History of Plant Tissue Culture 1. The concept of plant cell totipotency - the ability of a single plant cell to autonomously produce a whole new plant - was first proposed by Gottlieb Haberlandt 2. in In 1902. 1937, Phillip White established the first successful root culture of tomato plants, demonstrating the viability of growing plant tissues in artificial media. 3. In 1941, Joannes Vanoverbeek used coconut milk to promote the growth and development of young Datura embryos, an early breakthrough in plant tissue culture 4. techniques. In 1957, Folke Skoog and Carlos Miller identified the critical role of auxin and cytokinin plant hormones in regulating root and shoot formation in tobacco tissue 5. cultures. In 1962, Toshio Murashige and Folke Skoog introduced the MS medium, a widely used nutrient formulation for plant tissue culture, particularly for tobacco. 6. In the early 1960s, Sanford Gurha and Dhruba Maheshwari obtained the first haploid plants through anther culture of the Datura plant, an important milestone in genetic engineering. Major areas of plant cell and tissue cultures and some fields of application Various Techniques of Plant Cell and Tissue Cultures Callus Culture Cell Suspension Protoplast Culture Anther Culture Callus is an Cell suspension Protoplasts are Anther culture undifferentiated cultures involve plant cells with involves growing mass of plant cells the growth of their cell walls pollen-producing grown in a single cells or removed. This cells from anthers nutrient-rich small cell clusters technique allows to generate medium. This in a liquid growth for genetic haploid plants. technique allows medium. This manipulation and This is a valuable for rapid enables efficient fusion, leading to tool for plant multiplication of mass production of the creation of new breeding and cells and can be cells for various plant varieties. genetics research. used to regenerate applications. entire plants.  Various techniques of plant cell and tissue cultures Callus Culture Cell Suspension Protoplast culture Anther culture Callus Multiplication Callus Initiation Callus Subculturing Callus Differentiation Callus is an unorganized Callus cultures are Depending on the plant mass of actively dividing repeatedly subcultured by species and culture cells derived from plant transferring portions of the conditions, callus cells can explants such as leaves, callus to fresh medium at be induced to differentiate stems or roots. The callus regular intervals, usually 2- into organized structures can be further multiplied 4 weeks. This allows the such as shoots, roots or on a suitable culture callus to continuously somatic embryos. This medium to produce large proliferate and maintain its allows for the regeneration amounts of unorganized state. of whole plants from the undifferentiated cells. callus culture. Cell Suspension Culture culture of cells or cell aggregates (small clumps of cells ) in liquid media. Cell suspension culture Cell Suspension Defined Medium Controlled Rapid Flexibility Growth Multiplication Cell suspension The liquid cultures are Cells are Cell suspension medium and grown in a suspended and cultures can agitation allow defined liquid agitated in the rapidly multiply, for easy medium, liquid medium, making them manipulation of providing the allowing for useful for mass factors like pH, essential controlled and propagation of temperature, and nutrients and uniform exposure cells, production the addition of growth factors to to the nutrients, of secondary specific support cell leading to metabolites, and compounds to division and consistent growth genetic influence cell growth. and engineering growth and development. applications. behavior. Protoplast culture Culture of plant cells with their cell walls removed Organ Culture Culture of isolated plant organs such as anthers, roots, buds and shoots. Method in Plant Tissue Culture: Methods of Plant Tissue Culture: Stage 0: Nutrient Preparation Stage 1: medium of donor initiation preparation plant Stage IV: Stage II: Stage III: Acclimatizatio Multiplication Rooting n Preparation of plant tissue culture media Should contain all of the following components: Macronutrients Micronutrients Vitamin Amino acids/nitrogen supplements Source of carbon (Sucrose/sugar) Undefined organic supplements Growth regulators (Cytokinins/auxin) Solidifying agents Media Composition and Sterilization Macronutrients Micronutrients Essential elements like nitrogen, Trace elements like iron, boron, and phosphorus, and potassium that zinc that are required in smaller support plant growth and development. quantities. Growth Regulators Solidifying Agents Hormones like auxins and cytokinins Compounds like agar that provide a that control and direct the plant's semi-solid support for the plant tissues. growth.  Murashige and Skoog (MS) medium Minor salts (micronutrients)/ 1L Boric acid (H3BO3) 6. 2 mg/l Vitamins and organic Major salts (macronutrients)/ Cobalt chloride (CoCl2 · compounds/ 1L 1L Myo-Inositol 100 mg/l Ammonium nitrate 6H2O) 0.025 mg/l Ferrous sulfate (FeSO4 · Nicotinic Acid 0.5 mg/l (NH4NO3) 1,650 mg/l Calcium chloride (CaCl2 · 7H2O) 27.8 mg/l Pyridoxine · HCl 0.5 mg/l 2H2O) 440 mg/l Manganese(II) sulfate Thiamine · HCl 1.0 mg/l Magnesium sulfate (MgSO4 (MnSO4 · 4H2O) 22.3 mg/l Glycine 2 mg/l · 7H2O) 370 mg/l Tryptone 1 g/l (optional) Potassium iodide (KI) 0.83 Monopotassium phosphate Indole Acetic Acid 1-30 mg/ (KH2PO4) 170 mg/l mg/l l(optional) Potassium nitrate (KNO3) Sodium molybdate Kinetin 0.04-10 mg/l(optional) 1,900 mg/l. (Na2MoO4 · 2H2O) 0.25 mg/l Zinc sulfate (ZnSO4·7H2O) 8.6 mg/l Ethylenediaminetetraacetic acid ferric sodium (NaFe- 4 Stages of Plant Tissue Cultures Aseptic Techniques and Equipment 1 Laminar Flow Cabinets 2 Autoclaves Provide a sterile work Sterilize media, instruments, environment to manipulate and glassware at high plant tissues without temperatures and pressures. contamination. 3 Micropipettes Precisely measure and transfer small volumes of media, solutions, and plant materials. Contamination: A Persistent Threat 1 Microbial Invaders 2 Sterilization Challenges Bacterial and fungal Ensuring thorough surface contamination can quickly sterilization of explants is overtake bamboo cultures, essential, but can be difficult leading to loss of explants and due to the hardy nature of failed propagation attempts. bamboo tissues. 3 Aseptic Technique Importance Strict adherence to aseptic procedures during all stages of the tissue culture process is crucial to prevent contamination. Culture Medium Optimization Medium Composition Role of Carbohydrates Phytohormones MS (Murashige and Carbohydrates like Cytokinins promote Skoog) medium is sucrose provide energy, axillary shoot growth, commonly used for carbon, and osmotic while auxins like IAA and bamboo shoot initiation. support during in vitro IBA can be inhibitory. Liquid media can culture. enhance shoot multiplication compared to semi-solid. Supplemental Additives 1 Coconut Milk 2 Antioxidants Increased multiple shoot induction Ascorbic acid, citric acid, and from nodal segments of seedlings cysteine enhanced multiple shoot and mature culms. induction in some bamboo species. In Vitro Rooting Seedling-derived Shoots Rooting is easier from shoots of seedling origin compared to older plant sources. Endogenous Auxins The presence of natural auxins is important, though exogenous application can also induce rooting. Rooting Frequency Rooting ability tends to decrease with increasing age of the source plant material. Acclimatization and Field Transfer Direct Acclimatization Potting Media Hardening In some cases, in vitro Healthy rooted plantlets Plantlets are maintained plantlets can be directly are transferred to various under high humidity transferred to greenhouse soil, sand, and compost before gradual transfer to conditions. mixtures. field conditions.