Transcript Lecture 2
Dr Maged El-Sayed Mohamed [email protected] [email protected] Course outline • Definitions • Culture environment Physical factors Growth medium Plant growth regulators • Culture types • Plant regeneration • Micropropagation Summary of the last lecture B: The growth medium Essential elements or mineral ions Organic supplement Source of carbon Gelling Agent a. Agar • Composition: 1. Agarose 2. Agaropectin • Agar tertiary structure • Advantages • Disadvantages b) Agarose c) Gelrite™ d) Phytagel™ C. Plant growth regulators: Definition Characteristics Auxin Cytokinin Gibberelin Classification of PGRs Abscisic acid Ethylene Other Jasmonates Salicylic acid Brassinosteroids Course outline • Definitions • Culture environment A.Physical factors B. Growth medium C. Plant growth regulators • Culture types • Plant regeneration • Micropropagation Culture Types • Explants • Types of explant: a) Root tip b) Shoot tip c) Embryo d) Haploid tissue A. Callus: • Definition: It is an unspecialized and unorganized, growing and dividing mass of cells,. • During callus formation there is some degree of dedifferentiation both in morphology and metabolism, resulting in the lose the ability to photosynthesis. • Habituation: • Compact callus • Friable callus B. Cell-suspension cultures • When friable callus is placed into the appropriate liquid medium and agitated, single cells and/or small clumps of cells are released into the medium and continue to grow and divide, producing a cell-suspension culture. • The inoculum used to initiate cell suspension culture should neither be too small to affect cells numbers nor too large too allow the build up of toxic products or stressed cells to lethal levels. • Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering. C. Protoplasts Definition: They are plant cells with the cell wall removed. Source: from either leaf mesophyll cells or callus or cell suspensions. Production : Removing the cell wall is achieved: 1. Mechanically: by Scissors and forceps, but low yield due to damaged cells 2. Enzymatically: Cell wall is removed using degrading enzymes (cellulase and pectinase) in a simple salt solution with a high osmotic potential to maintain the cells. • Liquid medium is not agitated so it must be shallow enough to allow aeration in the absence of agitation. Applications: 1. Ideal target for transformation and genetic engineering. 2. Can be developed to callus on solid medium 3. Used for ultra-structure studies of plant cells 4. Isolation of subcellular components such as nuclei and chromosomes D. Hairy root cultures: Definition: • It is the culture produced after the infection of explants or cultures by the gram negative soil bacterium Agrobacterium rhizogenes. • This processes take advantage of the naturally occurring hairy root disease in Dicotyledons. Break Production of hairy roots in vivo: a) Agrobacterium recognizes some signal molecules exuded by wounded plant cells and becomes attached to it. b) The bacteria contain the Root inducing plasmid (Ri-plasmid) c) The bacteria genetically transfer part of the Ri-plasmid called the transfer DNA (T-DNA) to the plant genome, where it is get expressed and make the plant cell to: 1. Proliferate by increasing the rate of cell division (cytokinie expession) and cell elongation (auxin expression) to produce the hairy roots. why? 2. Produce the opines which is a type of unusual amino acids (octopine, agropine,nopaline, mannopine, and cucumopine) which is used by the bacterium as a carbon, nitrogen and energy source Agrobacterium cell Ri-plasmid Plant cell Structure of Ri-plasmid Ri-Plasmid Induction of hairy root cultures in vitro: 1. Explants are wounded and then inoculated with Agrobacterium rhizogenes. 2. Usually two or three days later, the explant can be transferred into solid media with antibiotics, such as cefotaxime, vancomycin or ampicillin to kill or eliminate redundant bacteria. 3. The hairy roots will be induced within a short period of time, which varies from one week to over a month depending on different plant species. 4. The decontaminated hairy roots can be subcultured on phytohormone-free medium. Advantages of hairy root cultures: 1. The hairy root system is genetically and biosynthetically stable 2. High production of secondary metabolites 3. The culture can grow under phyto-hormone-free conditions. 4. The culture shows fast growth which reduce the culture time and easy the handling Application of hairy root cultures: 1. Functional analysis of genes 2. Expressing foreign proteins 3. Production of secondary metabolites 4. The culture may Produce compounds which is not found in untransformed roots 5. The culture may change the composition of metabolites 6. The culture can be used to regenerate a whole plants Course outline • Definitions • Culture environment A.Physical factors B. Growth medium C. Plant growth regulators • Culture types • Plant regeneration • Micropropagation Plant Regeneration Somatic embryogenesis Organogenesis A. Somatic embryogenesis Definition: A process in which embryo-like structures are formed from somatic tissues and developed into a whole plant. Types: 1.Direct somatic embryogenesis: • The embryo is formed directly from a cell or small group of cells such as the nucellus, styles or pollen without the production of an intervening callus. • Direct somatic embryogenesis is generally rare 2.Indirect somatic embryogenesis • Callus is first produced from the explant and then embryos are produced from the callus tissue or from a cell suspension cultures. Indirect somatic embryogenesis in carrot (dauctus carota) 2,4 D (1 mg/L) Indirect Somatic embryogenesis Abscisic acid (0.025 mg/L) B. Organogenesis Definition: The production of organs, either directly from an explant or from a callus culture. • Organogenesis depend on adventitious organs arising either from a callus culture or directly from an explant or on the formation of axillary bud to regenerate whole plants from some types of tissue culture. Summary • Protoplast cultures • Hairy root cultures • Plant regeneration: 1. Somatic embryogenesis 2. Organogenesis