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83 Plant Tissue Culture Text Pages: 643 – 649; 659 – 673. Objectives: 1. Be able to describe and explain terms involved in plant tissue culture. 2. Be able to describe and explain different types of plant tissue culture as they relate to the type of plant part(s) regenerated. 3. Be able to distinguish and identify which type or technique of plant tissue culture should be used based on the type of propagation desired. I. PLANT TISSUE CULTURE A. Terms: 1. Tissue culture - is the science of growing plant cells, tissues, or a. Biotechnology – what is it? b. Biotechnology is the use of 2. Aseptic - free from microorganisms: a. Tissue culture is completed under b. Microbes may or may not 3. Explant – the plant part that a. An explant can be a single cell, tissue, b. The explant should also be free from 4. Micropropagation - is the production of plants from 84 a. Micropropagation involves the use of single cells, a tissue, or b. For this class, we are interested in multiplying whole plants 5. Somatic - cells or tissues that are 6. Germ - cells or tissue that are involved in 7. Embryo - is a rudimentary plant 8. Propagule - tissue that is divided and used 9. Plantlets - small complete plants that were B. Definitions for Plant Tissue Culture - Complete List: Adventitious - in reference to a bud, embryo, root or shoot that arises in tissues and locations that are not the normal origin in the plant. Aseptic - free from bacteria, fungi or other microorganisms. Autoclave - equipment that provides heat under high steam pressure for purposes of sterilization. Axillary bud or shoot - a bud or shoot that arises from the axil of leaves or normal origin. Callus - tissue that develops as a response to injury caused by physical or chemical means; cells may be differentiated but are unorganized. Cell - a structural and physiological unit of a living organism (plant). DNA (Deoxyribonucleic acid) - composed of organic chemicals and is the genetic material that ultimately determines an organism's characteristics. Embryo - a rudimentary plant 85 Epigenetic changes - persistent changes in phenotype that involve the expression of particular genes. Explant - the plant part that is put into tissue culture. Gene - specific sequence of DNA that codes for a specific trait. Genotype - the sum total of all genes present in an organism (plant). Germ - cells or tissues that are involved in reproduction and have one-half of the genetic material of somatic cells or tissues. Habituation - tissue cultures losing their requirements for a supply of exogenous growth regulators. In vitro - isolated from the living organism and artificially maintained. Micropropagation - the production of plants from plant parts used in tissue culture. Mutant - an organism (plant) that has a mutation. Mutation - change in the DNA sequence that is different from the original sequence. Organ - a distinct and visibly differentiated part of an organism. Organogenesis - refers to forming organs, in particular both roots and shoots from callus cells or other types of cells (e.g., parenchyma cells). Phenotype - actual appearance and behavior of an organism (plant). Plantlets - small complete plants that were produced via tissue culture. Propagules - tissue that is divided and used for further multiplication. Somaclonal Variation - variation (usually in phenotype and/or perhaps genotype) induced in cells by the tissue culture process. Somatic - cells or tissues that are vegetative and contain the complete genetic material of the organism (plant). Subculture - divide a propagule and transfer individual parts into other culture vessels. Tissue - a group of cells organized into a structural and functional unit. Tissue culture - is the science of growing plant cells, tissues, or organs under artificial conditions. 86 C. Types of Tissue Culture – Based on Type of Regenerated Plant or Part 1. Many types of tissue culture can be used to reproduce plants, a. The techniques described will be based on b. Almost all techniques could be used to regenerate whole plants, but some 2. Seedling formation - a. Aseptic seed culture - used for plants, such as i. culture medium keeps seeds alive ii. in nature, orchid seeds develop with the help of b. Embryo culture - involves excising an i. embryo rescue - the technique is used to rescue or save embryos ii. ovule culture and ovary culture are also methods of embryo culture 3. Plantlet formation a. A plantlet is b. Plantlet formation is divided into two categories: c. Axillary shoot formation i. where is the axil? 87 ii. two types of axillary shoots formation will be covered: iii. meristem culture - uses the meristem tip aa. the apical meristem is removed (excised) from bb. the explant usually is less than cc. this method is usually used to obtain dd. viruses and other pathogens are absent from the newest cell layers and ee. Rule to Remember: The smaller the amount of tissue taken, ff. meristem culture is successful for iv. Axillary shoot cultures - are techniques that involve stem aa. these techniques are usually referred to as micropropagation and used extensively in bb. because the apical meristems are already formed, these techniques provide reliable cc. with these techniques, lateral (axillary) buds break and dd. large numbers of lateral shoots often grow, allowing ee. the techniques designated as axillary shoot cultures include: i. axillary branching - many shoots form from 88 ii. nodal cultures - single nodes are cut from iii. 'stool' shoots - a shoot with several nodes in laid at an angle aa. what causes numerous stems to grow from a axil? bb. answer: d. Adventitious shoot formation – can lead to i. shoots are considered organs, so adventitious shoots are aa. direct shoot formation – shoots form directly bb. indirect shoot formation – callus forms first on explant and ii. roots also form via organogenesis iii. species, explant selection, and growth regulators used can iv. explants include almost any plant part: v. high rates of regeneration can be obtained vi. increased numbers of shoots with different or e. Regenerate haploid plants - i. plant part characteristic: ii. two techniques: 89 iii. either an anther or immature pollen is isolated from flowers 4. Callus formation – a. Stationary callus cultures – i. callus – response from ii. callus (wound cells) grow/proliferate and iii. high auxin concentrations are often used to promote callus formation, but iv. callus can be used for organogenesis b. Cell suspension culture - is callus tissue placed in liquid culture medium, i. whole plants can be regenerated ii. this technique is usually used for research c. Protoplast culture - are plant cells without i. cell walls are ii. this technique is usually used for research, 5. Somatic embryogenesis a. Embryogenesis - refers to forming an underdeveloped or 90 b. Somatic - refers to the embryo forming from a c. Somatic embryos can be formed by d. To be considered somatic embryos, the developing embryos must go through C. Summary 91 Micropropagation of Plants Text Pages: 650 – 659; 699 – 702; 712 – 723. Objectives: 1. Be able to describe and explain the advantages and liabilities of micropropagation. 2. Be able to name and identify the four stages of micropropagation. 3. Be able to describe and explain the factors that affect establishment of explants in tissue culture (Stage I). 4. Be able to describe and explain the factors that affect shoot multiplication during micropropagation (Stage II). 5. Be able to describe and explain the various manipulations used to promote rooting during Stage III of micropropagation. 6. Be able to describe and explain how a plantlet changes during Stage IV of micropropagation. 7. Be able to predict how various factors involved in the different stages can affect the success of micropropagation. II. MICROPROPAGATION A. Introduction 1. Micropropagation is an important propagation technique for 2. Microshoots, in vitro grown shoots, are used in micropropagation and a. Axillary buds – b. Organogensis or adventitious shoot regeneration – 3. Micropropagation is the term used for in vitro production of plants, 4. For this course, micropropagation involves axillary shoot cultures 92 B. Characteristics of Micropropagation 1. Advantages of micropropagation a. Mass production of specific clones i. very large numbers of plants can be produced ii. plantlets take much less space iii. rapid multiplication is important for bringing large numbers of new b. Year-round plant (nursery) production c. Production of pathogen-free plants i. micropropagation can be used to eliminate ii. aseptic conditions must be d. Clonal propagation of clonal stock for seed production e. Germplasm preservation 2. Disadvantages or problems with micropropagation a. Facilities required are costly b. Workers must have certain skills and c. Multiply errors: plant identity, introduction of a pathogen or 93 d. Genetic or epigenetic changes caused by III. STAGES OF MICROPROPAGATION A. Introduction - Four Stages of Micropropagation (Axillary Shoot Proliferation) 1. Establishment and Stabilization – of 2. Multiplication – of 3. Root Formation (also considered Transplant preparations) 4. Acclimatization – 5. The importance of these stages varies B. Stage I - Establishment and Stabilization of the Explant 1. Explant disinfestation – surface sterilize an explant a. The explant must be free from all b. External surfaces and internal tissues must be c. Ideally the tissue from which the explant is removed should be in d. Keeping the foliage, including stems, dry 2. Explant selection is very important a. Ontogenetic state – 94 b. The plant part used as the explant should be selected to c. Plants resulting from micropropagation should be true-to-type without d. Repeated subculturing – may help explant shoots and propagules to become 3. Culture medium requirements – a. The basic medium contains i. woody plants were at first difficult to culture because ii. a medium lower in b. Growth regulators must be applied i. usually concerned with ii. these plant growth regulations must be applied in 4. Environmental parameters during culturing must be regulated too a. Light levels and b. Temperature should be kept between 5. Exudation – endogenous plant substances a. Some of these substances can be washed away 95 b. Ways to limit explants from producing these inhibitors is to use antioxidants 6. Stabilization – explants should be stable in vitro, meaning microshoot growth a. The establishment stage usually lasts b. Woody plants often take longer to stabilize than C. Stage II - Multiplication of Propagules 1. The objective of this stage is to increase a. Subculturing - means taking in vitro cells, tissues, or organs, microshoots in the case of micropropagation, dividing them, and putting b. Take home message: consider both the number of subcultures and the shoot 2. Multiplication of shoots a. Propagation ratio - i. number of shoot produced can vary ii. optimize the nutrient medium to obtain b. Growth regulator concentrations i. usually use a high cytokinin to auxin ratio (e.g., 100:1) ii. the ratio is not absolute and must be 96 3. Frequency of transfer (subculturing) a. The time needed between transfers (subculturing) may vary b. If transfer (subculturing) is delayed, the propagules may c. Example: D. Stage III - Root Formation: preparing the microshoot for 1. Quality of the microshoots a. Quality and health are influenced by b. Strong, healthy microshoots should be c. Stage III can be skipped if 2. Growth regulators are shifted to a. Cytokinin concentration is b. Auxin level should be regulated c. A high cytokinin concentration used in Stage II can inhibit 3. Other medium components are also shifted, including a. Salts or minerals may b. Agar content may be increased to stiffen 97 4. During or after root formation, plantlets must be "toughened-up" a. Plantlets are susceptible to problems due to b. Problems include: E. Stage IV - Acclimatization: transplanting the plantlet into 1. The "toughened" plant is transferred from 2. The plant must now support itself or a. Under tissue culture conditions, the propagule depended b. Green leaves on microshoots may or may not 3. The plantlets must adjust a. Plantlets must become b. The plantlet must also acclimate to 4. Stomata on leaves of tissue cultured plantlets a. Stomata stay open so that plantlets transpire at high rates b. The type of cytokinin used c. Therefore, growth regulator uses during micropropagation can (1) affect explant 98 5. Plantlets should be protected from pathogens during Stage IV since a. Proper sanitation must be used including b. Minimize plant stress F. Summary c:\plsc300\12notes-9.doc