Plant Tissue Culture

1. Plant Tissue Culture Dr Maged El-Sayed Mohamed memohamed@zu.edu.eg maged789@hotmail.com

2. Summary of the last lecture

3. 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: • Mechanically: by Scissors and forceps, but low yield due to damaged cells • 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.

5. Hairy root cultures: • Definition: • It is the culture produced after the infection of explants or cultures by the gram negative soil bacterium Agrobacteriumrhizogenes. • This processes take advantage of the naturally occurring hairy root disease in Dicotyledons.

6. Plantcell Agrobacterium cell Ri-plasmid

7. Structure of Ri-plasmid Ri-Plasmid

8. Induction of hairy root cultures in vitro • Advantages of hairy root cultures • Application of hairy root cultures

10. Indirect somatic embryogenesis in carrot (dauctus carota) 2,4 D (1 mg/L) Indirect Somatic embryogenesis Abscisic acid (0.025 mg/L)

11. Course outline • Definitions • Culture environment • Physical factors • Growth medium • Plant growth regulators • Culture types • Plant regeneration • Micropropagation

12. Micropropagation • Definition: Rapid clonal in vitro propagation of plants from cells, tissues or organs cultured aseptically on defined media under controlled conditions of light and temperature. • The asexual or vegetative propagation (multiplication) of plants in vitro • Applications: • Propagation of large numbers of uniform plants. • May allow faster production of plants that are slow to propagate in vivo. • It may decrease the time needed for bulk-up of new cultivars before they are introduced commercially.

13. Stages of Micropropagation Micropropagation is divided into 5 stages. Stage 0: Preparative Stage: • Donor plant selection and explant preparation • Explant quality is influenced by the physiological condition of the donor plants. • The explant should be Pathogen free and maintained in clean conditions.

14. Stage 1: Establishment of explant in culture: • Surface-sterilization of explant tissues. • Isolation of explant under sterile conditions. • Medium must contain all components necessary to make the explant perform as desired (medium composition and PGRs). • Adjust the environmental conditions (Light, Temperature, Relative humidity, etc)

15. Stage 2: Multiplication and proliferation of axillary shoots • Repeated enhanced shoot production by high cytokinin in the medium, alone or with a smaller amount of auxin. • Shoots must be transferred to fresh medium at regular intervals. • Number of subcultures possible from the original culture varies with species/cultivar.

16. Stage 3: Pre-transplant (Rooting) • It is rooting of shoots or shoot clusters in vitro. • Auxins are important for root initiation in vitro • Advantages of rooting after removal from culture • Reduced costs • Structurally and physiologically better • Avoid damage to roots that occur during transplanting

18. Stage 4: transfer to natural environment: • Acclimatization: a process of physiologically and anatomically adjustment from in vitro to ex vitro conditions. • Relatively slow process, may take weeks. • Must adjust from high to lower relative humidity (e.g. from 98-99% to 20 - 60%): development of sufficient defenses to control water loss. • Must adjust from low light to high light: from low photosynthetic competence (heterotrophic nutrition) to photosynthetic competence.

20. Advantages of Micropropagation: • From one to many plants rapidly • Multiplication in controlled lab conditions • Continuous propagation year round • Potential for disease-free plants

21. Limitation (Disadvantages) • Equipment/facility intensive operation • Technical expertise in management positions • Protocols not optimized for all species • It may be too expensive

23. Break

24. General plant tissue culture laboratory design • Glassware washing and storage area • Media preparation and sterilization area • Refrigerator/freezer • Balances • Hot plate/stirrer • Ph meter • Autoclave • Growth room • Aseptic transfer area • Laminar flow hoods

27. Sterilization methods: • Why? • Micro-organism contamination can over grow the plant culture resulting in culture death • Micro-organism contamination exhaust the nutrient media • Micro-organism can change in secondary metabolite structure or produce other compounds . • What? • The explant or culture • The vessels The media The instruments • The environment where handling is taking place

28. How? Heat sterilization: a. Dry heat: 130-180 °C for 2-4 hours Used for glassware, metal instruments Autoclave: 121 °C, 1.06 kg/cm2 (15 Ib) for 15 to 20 minutes Used for glassware, media and aqueous solutions and plastic caps Sterilization by filtration: for heat labile aqueous solutions Ethanol: used for handling surface sterilization and also for explant. Chemical sterilization: using sodium or calcium hypochlorite, sliver nitrate, mercuric chloride or some other bactericidal chemicals

29. Secondary metabolites production • Secondary metabolites could be produced from culture in amounts more than or less than the original plant and this depend on: • Origin of tissue • Physical factors (pH, temperature etc) • Media formulation (carbon source, PGRs and other elicitors)

30. Elicitation Difinition: an Elicitor may be defined as a substance which, when introduced in small concentrations to a living cell system, initiates or improves the biosynthesis of specific compounds.

31. Classification of Elicitors A) Biotic elicitors. From biological origin They include: 1. Enzymes and polysaccharides derived from microorganisms 2. Phytochemicals produced by plants in response to physical damage, fungi or bacteria attack,

32. Classification of Elicitors B) Abiotic elicitors are the substances of non-biological origin. 1. Chemicals such as inorganic salts, heavy metals, some chemicals that disturb membrane integrity. 2. Physical factors like mechanical wounding, ultraviolet irradiation, high salinity, high or low osmolarity, extreme temperature (freezing, thawing), high pressure.

33. Thank you Dr Maged El-Sayed Mohamed memohamed@zu.edu.eg maged789@hotmail.com