Lecture-1.pdf

Plant cell and Tissue culture (PHCG 433 E) PG E08 Dr- Osama Elsabagh Lecture-1 Introduction  PG E08 (2 cr) PHCG 433E / PHCG 433E. (3 cr) Semester Work 25 Mid Term 25 Quiz 20 Ora...

Plant cell and Tissue culture (PHCG 433 E) PG E08 Dr- Osama Elsabagh Lecture-1 Introduction  PG E08 (2 cr) PHCG 433E / PHCG 433E. (3 cr) Semester Work 25 Mid Term 25 Quiz 20 Oral 15 Practical 45 Final 60 Oral 15 Final 45 Total 100 Total 150  What is Plant Tissue Culture? It is a form of asexual propagation of plant protoplasts, cells, tissues or organs under aseptic laboratory conditions which lead to cell multiplication or regeneration of organs or whole plants. What is Protoplasm? Protoplasm is the living part of the cell, which comprises of different cellular organelles. It is a jelly-like, colourless, transparent and viscous living substances present within the cell wall 07/21/2024 5 Experimental systems based on plant cell and tissue culture are characterized using isolated parts of plants, called explants, obtained from an intact plant body and kept on, or in a suitable nutrient medium (May be either semisolid, such as agar, or liquid, such as purified water). This nutrient medium functions as replacement for the cells, tissue, or conductive elements originally neighboring the explant.  Plant Cell and Tissue culture Tissue culture is defined as in vivo septic cultivation of isolated plant organ or tissues. X  In-vitro aseptic cultivation of isolated plant organs or cells on a nutrient medium (roots, shoot-tips, leaves, embryos.)  In-vitro: in glass  Aseptic: free from microorganisms  Plant cells are totipotent IF plant can't grow by asexual pross =nontotipotency Totipotency: ability of a cell, tissue or organ (non-embryonic) to grow and develop (like that of a zygote) leading to the formation of a new entire plant identical to the original Overview of the tissue culture process   Plants cultured in vitro yield thousands of genetically identical plants (clones) from a single plant. This process is called micro-propagation and is used to commercially propagate plants asexually. The Purpose of Tissue Culture what is goal of tissue culture ? Bioactive compounds extracted from plants are widely used. The natural habitats for many plants are rapidly destroyed leading to extinction (disappearing) of many valuable and even endemic species.  When to perform plant tissue culture? If the source plant: 1. Is difficult to cultivate. 2. Has a long cultivation period. 3. Produces a commercially significant compound that cannot be chemically synthesized in large scale. 4. Has a low metabolite of the compound of interest. Secondary metabolites can be produced by plant cell and tissue culture techniques under controlled and reproducible conditions, independent of geographic and climatic conditions.  Uses and Importance of plant tissue culture A- Practical applications in agriculture:  The production of disease-free plants. Haploid is the presence of a single set of chromosomes in an organism's cells. Sexually reproducing organisms are di-haploid  Production of di-haploid plants from haploid cultures to achieve homozygous lines more (having two sets of chromosomes, one from each parent). In humans, only the egg and sperm cells are haploid. rapidly in breeding (reproduction) programs.  Genetic transformation: introduces the foreign DNA to generate novel (and typically desirable) genetic combinations by the expression of genes.  Cryopreservation: Store valuable plant germ plasm in culture under low temperature. B- Medicinal importance: 1- Large-scale growth of plant cells in liquid culture as a source of secondary products. 2- Discovery of new metabolites. 3- Selection of superior strains of medicinal plants. 4- Development of commercial production of expensive bio medicaments. 5- Elucidation of biosynthetic pathways of secondary metabolites with isolation of corresponding enzymes. 6- Improvement of medicinal plant species by genetic engineering. C- Pharmaceutical industrial importance  Pharmaceutical industry can’t depend on conventional botanical sources of drugs due to the following factors:- 1. Availability of raw material: Plants although having bio-medically active ingredients they can’t be produced in an economically enough to safety demand.  e.g. Taxol (Anticancer drug from Taxus brevifolia)  100 years old tree of Taxus is suitable for collection of trunk or stem bark.  Bark of 3 matured tree would give 1g of Taxol.  Generally, 2 g Taxol is required for cancer treatment. 2. Fluctuation of supplies & quality:  Crude drug yield fluctuate due to: Climate –crop diseases-varying methods of collection & drying –inherent variation of active constituents arising from plants of the same species but are different genetically. 3. Political considerations:  When a new drug has been successfully marketed, its country of origin may put limits on the export of the crude drug & process the plant for the active constituents.  e.g.: Opium Poppy cultivation & trade. 4. Patent rights:  It is not possible to patent naturally occurring plant metabolites (only the method for its extraction & isolation).  So, no company will invest in study & formulation for a plant for which it will have no patent rights. Accordingly; plant tissue culture affords the following:  Production at all times could be geared to demand.  Product of standard quality assured.  Highly complicated & specific method of production can be patented.  Explant (differentiated cells) Callus (Undifferentiated cells) meristemoid Parenchyma cells primordial organ Tissue Culture: De-differentiation to Regeneration The phenomenon of a mature cell reverting back to meristematic state to form undifferentiated callus tissue is called de-differentiation. Micropropagation  In vitro propagation of plants vegetatively by tissue culture to produce genetically similar copies of a cultivar is referred to as micropropagation or clonal propagation.  Micro-propagation Methods: (I) Organogenesis: Is a process involving re-differentiation of meristematic cells present in callus into shoot buds. These shoot buds which in turn give rise to leaf primordial and the apical meristem. (II) Somatic embryogenesis: Is a process involving redifferentiation of meristematic cells into non-zygotic somatic embryo which are capable of germinating to form complete plants. Any cell of the female tissue around the embryo sac may give rise to an embryo and such embryos are called non-zygotic embryos.  Sources & Isolation of Tissue Explants  Tissue cultures can be derived from many parts of the plant (explant).  For example, pith tissue, root tips, shoot tips, embryo, anther and young leaves.  Surface of the explant is free from contaminating micro-organisms.  Explants: mature, differentiated tissues removed from a plant.  Callus: is a largely unorganized mass of parenchyma cells, it is initiated and maintained on nutrient media in vitro, the cells contain all genetic information present in the normal plant. Its formation marks the beginning of successful plant cell culture. Factors taken in consideration in selecting the explants  1- Part or organ of the plant must have meristimatic cells.  2- Age of the plant: young plant usually provides the best explant due to: A- Higher meristimatic tissues B- No reproductive structure formation. C- less contamination  3- Explant size: several small explant cultured in the same vessel grow faster than large explant cultured alone N.B. (large explants suffer from higher contamination).