Lecture 4- Tissue culture.pdf

Lecture 4 Plant Tissue culture BIOC 3260: Principles of Biotechnology Dr. Angela T. Alleyne 1 LEARNING OUTCOMES At the end of this lecture you will be able to: 1. Review the history of plant tissue culture (PTC) 2. Differentiat...

Lecture 4 Plant Tissue culture BIOC 3260: Principles of Biotechnology Dr. Angela T. Alleyne 1 LEARNING OUTCOMES At the end of this lecture you will be able to: 1. Review the history of plant tissue culture (PTC) 2. Differentiate between solid and liquid culture methods 3. Discuss the use of different plant tissue culture methods 4. Explain the use of auxins and cytokinins in culture 5. Define some important TC terms, e.g. terms plasticity, totipotency, explant, callus 6. Explain the general steps in tissue culture 7. Discuss the uses of plant tissue culture 2  Why PTC? After plant transformation, whole plants (clones) are required from isolated plant cells or tissues that were transformed. Must be done in vitro so that a high regeneration frequency is achieved Note: high regeneration frequency does not necessarily mean high transformation frequency 3  Applications of PTC http://www.biocyclopedia.com/in dex/images/Biotechnology/chapte r09/079_large.jpg 4  Plant Tissue culture(PTC) White (1939) defined plant tissue culture as a system in which cells satisfied two main requirements of remaining ‘‘undifferentiated yet capable of unlimited growth’’ (White, 1939). Plant tissue culture (PTC) is a term describing techniques used to propagate plants vegetatively by using small parts of living tissues (explants), on defined artificial growth media with under sterile conditions. while Micropropagation is the production of whole plants through tissue culture from small parts such as shoot and root tips, leaf tissues, anthers, nodes, meristems and embryos. 5 Brief history 1902-(meristem cultures) a German physiologist, Gottlieb Haberlandt developed the concept of in vitro cell culture. He isolated single fully differentiated individual plant cells from different plant species and pointed out the possibilities of the culture of isolated tissues. 1934-1939-( in vitro embryo culture) White established continuous callus culture while working on cell metabolism. Introduced vitamins to add to tissue cultures. 1957-(organogenesis) Skoog and Miller- Skoog and Miller worked further to propose the concept of hormonal control of organ formation (1957). Their experiment on tobacco pith cultures showed that high concentration of auxin promoted rooting and high kinetin induces bud formation or shooting. 6 Brief history 1962 Murashige and Skoog prepared a medium by increasing the concentration of salts This media enhanced the growth of tobacco tissues by five times. Today MS medium is the most widely used media abase in tissue culture. They also demonstrated the use of auxin and cytokinin in tissue culture media 1960 Thorpe, T. A. (2007). History of plant tissue culture. Molecular Biotechnology, 37(2), 169-80. Isolation and regeneration of protoplasts first demonstrated by Prof. doi:https://doi.org/10.1007/s1 Edward C Cocking 2033-007-0031-3 1980’s-present Protoplast fusion and the development of somatic hybrids. This period onward led to many applications of TC to enhance plant breeding efforts. 7 Stem cells have the ability to differentiate into. any type of cell Totipotency and plasticity 1. The ability of a cell or tissue or organ to grow and develop into a fully differentiated organism ( whole plant in this case) given the correct stimulus. 2. The ability of a cell to differentiate into any cell type of an organism (Condic 2014). Genetic potential is maintained. 3. Plasticity- ability of plant cells to adapt to changing environments through altered metabolism. 8  Any portion of leaf, shoot, Explant stem, root, or living plant parts used for regeneration in tissue culture Younger more rapidly growing tissues make better explants Usually transferred to artificial media and grown in hormones to facilitate differentiation 9  Components of a tissue culture medium Three basic components 1. Essential mineral elements- usually a complex salt mixture Macronutrients- 25-60 mM of inorganic nitrogen is satisfactory for plant cell growth Micronutrients- Plant Growth regulators ( PGR’s) 2. An organic base- supplies amino acids and vitamins 3. Fixed carbon – usually sucrose * TC media contains approximately 95% water 10 Callus culture- usually associated with use of solid TC media Suspension culture- associated with liquid TC conditions Taken from Clark Biotechnology 11  Types of tissues Protoplast cultures- cell Embryo Callus- used Cell Microspore walls removed, Shoot tips and culture- to initiate cell suspension - usuallay culture- use of Meristem Root culture- embryos may suspension liquid culture isolated from haloid pollen culture- young root be used to cultures, of friable leaf tissue or or anthers as culture of used as generate densely callus , cell explants. suspensions. apical shoot explants callus or aggregated or maintained as Used in plant Cell wall can be tips somatic friable batch cultures breeding removed embryos mechanically via enzymes 12  Summary of PTC methods What is included ?  Typical Media composition (MS) Macronutrients Micronutrients Carbon source Organic supplements PGR’s N- KNO3 and NH4NO3 Fe- FeSO4. 7H2O Sucrose Thiamine ( Vitamin Auxins- 2,4-D, IAA, B1) IBA, NAA P- KH2PO4 Mn- MnSO4. 4H2O Myoinositol (vitamin Cytokinins- BAP, B) Kinetin, Zeatin K- KH2PO4 Co- CoCL2. 6H2O Amino acids e.g- Abscisic acid glycine, casein. Mg- MgSO4. 7H2O Zn- ZnSO4. 7H2O Ca- CaCL2. 2H2O Cu- CuSO4. 5H2O S Mo- NaMoO4. 2H2O B- H3BO3 14  Differentiation and dedifferentiation Organogenesis in plant tissue culture involves two distinct phases: dedifferentiation and redifferentiation. Dedifferentiation -begins shortly after the isolation of the explant tissues. Mature cells have accelerated cell division and form a mass of undifferentiated cells (called callus). Cellular events, that allow cells to divide once again, are A change in chromatin organization termed dedifferentiation. A dedifferentiated tissue accompanies the dedifferentiation can act as meristem (e.g., vascular cambium, wound stage. meristem, cork cambium). Redifferentiation, also called budding dedifferentiated cells/tissue which lose the ability to divide in PTC, may begin any time after the are called redifferentiated cells/tissues and the event, first callus cell forms. redifferentiation. 15  Differentiation and dedifferentiation dedifferentiation increases the developmental potency of cell Transdifferentiation, developmental changes between cell lineages regardless of the Cellular events, that allow cells to divide once again, are cellular potency. termed dedifferentiation. A dedifferentiated tissue can act as meristem (e.g., vascular cambium, wound meristem, cork cambium). Dedifferentiated cells/tissue which lose the ability to divide are called redifferentiated cells/tissues. 16  Differentiation pathways a plant cell In PTC, transdifferentiation leading to increased developmental potency is often referred to as dedifferentiation, especially during callus formation. Source: Volume 10 - 2019 | https://doi.org/10.3389/fpls.2019.00536 Differentiation is generally associated with So, callus formation is the decreased,and dedifferentiation with increased results of trans of developmental potency. dedifferentiation 17  A mass of undifferentiated parenchyma type cells. Often performed in the dark. Callus Auxin= cytokinin in media Wound responses and callus formation had been observed and the term “dedifferentiation” was coined in the 1950s Callus formation is the result of overproliferation or transdifferentiation of differentiated cells. dedifferentiation is strongly associated with callus formation since callus is widely regarded as a proliferating mass of “dedifferentiated cell. 18  Organogenesis Process of initiating development of shoot or root primordia in vitro Can occur directly or indirectly Directly from the same organ type Indirectly through callus Organ Indirect: Explant callus Meristem primordia cells cells Direct: Organ Explant Meristem primordia cells cells 19  Regeneration of whole plants from Somatic embryogenesis cultured plant cells in only one step via embryo formation Process of initiation and development of embryos from somatic cells e.g., Plant protoplasts Direct or indirect Direct- use of leaf or other organ tissues as explants to produce embryos Indirect use of a callus phase http://slideplayer.com/slide/3467616/12/images/5/sequential+ stages+of+somatic+embryo+development.jpg 20 Direct and Indirect regeneration 21 Meristem tip culture 22  Plant Growth Regulators Auxins 2,4, dichlorophenoyacetic acid ( 2,4-D) indole acetic acid (IAA) - Napthylacetic acid (NAA) Cytokinin 6- Benzylaminopurine ( BAP) Kinetin- ( Ki) Zeatin This Photo by Unknown Author is licensed under 23 CC BY  Cytokinins Naturally occurring cytokinins have a substituted N6 terminus in adenine 24 Auxins 25 Auxin and Cytokinin ratios SHOOTS High auxin High and cytokinin Low and Low cytokinin auxin ROOTS 26  Tissue selection 0 Typical Tissue culture steps and sterilization Initiation of culture I in vitro Multiplication- sub-culturing of explant on shooting II media Rooting- explant grown III on rooting medium Hardening- Young IV plant grown in soil 27  Advantages Large volumes of mature plants can be produced in a short time for example this may allow fast propagation of new cultivars, Endangered species can be cloned safely, Large quantities of genetically identical plants can be produced, Plant production is possible in the absence of seeds, The production of plants having desirable traits such as good flowers, fruits and odor is possible, Whole plants can be regenerated from genetically modified plant cells, Disease-, pest- and pathogen-free plants in sterile vessels are produced and distributed for improved agriculture. 28 can lead to undesirable Somaclonal variation traits Many callus cultures grown indefinitely eventually undergo some type of mutation in culture Causes: biochemical, genetics, physiological This can be used to screen for unique biotypes and analyzed for plant breeding - Mutation plant breeding has been used with tissue culture to develop resistant plants, herbicide tolerant plants etc. Typically a traditional crop improvement cycle may take 10–15 years to complete e.g. through traditional breeding etc. TC and use of somaclonal variation may speed up this process 29 Epigenetics and PTC PTC are also affected by epigenetic changes exhibited at, e.g. histone methylation/demethylation In the membrane, the NADPH-dependent-oxidases respond to stress Stresses accompanying tissue culture plant regeneration affect the proper functioning lant organelles resulting in ROS. Under stress homeostasis is not preserved, and the excess of ROS may manifest in the degradation of pigments, proteins, lipids and DNA affecting cellular functioning Chloroplasts and peroxisomes are the primary sources of ROS under light conditions, while in the dark the mitochondria is the source of RO 30  Somaclonal variants Genetic differences from the same culture process which generates mutant phenotypes. Common genetic changes affecting the regenerants, include DNA sequence changes- SNPs, gene amplification, transposition, and chromosomal alterations. 31 Case study: Fusarium wilt resistance in banana Use of somaclonal variation in banana to generate plant resistant to Fusarium wilt. Taken from: Mol Biol Rep ( 2014) 41:7929-7935 32  Agricultural biotechnology pros 125 millions ha of transgenic crops in 2008 reduction of pesticides application transgenic plants with a better nutritional quality mass propagation of selected and healthy plants production of proteins for industrial or therapeutic use improved the competencies of the agricultural systems both in industrial and in developing countries- development of Biosafety regulations introduction of genetically modified organisms (GMOs) on the market are an important source of agricultural biodiversity. Better regulation of trade in agriculture products- SPS laws and regulations - Improved testing for use and commercialization of GMOs — whether plants, animals or microorganisms — new diagnostic techniques and kits. 33  Some ethical considerations 1. The risk of escapes- new genes introduced into plants could escape and be transferred to other plant species 2. Development of new viruses in transgenic plants 3. Increased genetic erosion (i.e. a reduction in biodiversity) 4. insect-resistant transgenic plants could lead to insect resistance and destroy beneficial insects 5. herbicide-resistant transgenic plants could induce selection of non- cultivated plants for resistance to herbicide 6. GMP development may lead to patent monopolies (patenting of genes and plants), which may in turn pose other moral problems. 34  References: Slater, A., Scott N.W. and Fowler, M.R. Plant Biotechnology: the genetic manipulation of plants. 2nd edition. Oxford University Press Thorpe, T. A. (2007). History of plant tissue culture. Molecular Biotechnology, 37(2), 169-80. doi:https://doi.org/10.1007/s120 33-007-0031-3 35