5_ Fiundamentals of Plant Cell Culture I..pdf

FUNDAMENTALS OF PLANT TISSUE CULTURE I  Plant Tissue Culture Plant tissue culture as modern biotechnology system, is becoming very important for the development of mankind, at present. Considered as one of the essential breeding approaches for many corps, vegetables and...

FUNDAMENTALS OF PLANT TISSUE CULTURE I  Plant Tissue Culture Plant tissue culture as modern biotechnology system, is becoming very important for the development of mankind, at present. Considered as one of the essential breeding approaches for many corps, vegetables and fruits Provides an alternative method for conventional vegetative propagation. Crops attained through tissue culture are established through time-saving and detailed approaches in comparison to traditional plant breeding. Reduce the life cycle of some species known to have relatively long-life cycle.  Plant Tissue Culture Totipotency based on the information that all plant cells except sperm and egg cells contain the full complement of genes, which makes it feasible to grow individual plant cells into full healthy plants that can be grown inside the laboratory or in vitro set up. This procedure is also called micropropagation. ▪ Capable of developing into complete organism or capable of differentiating into any cell type of an organism. Often misused as “ All/most plant cells are totipotent”  History 1902 The idea of the totipotency oof plant cell was given by Haberlandt 1937 White - first time established successful root culture of tomato 1941 Vanoverbeek used coconut milk for growth and development of young Datura embyros 1957 Skoog and Miller demonstrated the role of auxin and cytokinin on root and shoot formation in tobacco-tissue 1962 Murashige and Skoog introduced the medium for tobacco culture 1987 Isolation of Bt. Gene from bacterium Bacillus thuringiensis  Plant Tissue Culture Totipotency Differentiation, Dedifferentiation and Redifferentiation Differentiation: Differentiation is a process through which meristematic tissues undergo permanent change to form specialized cells in the plant body. Differentiation leads to the formation of permanent tissues which have specialized structures for specific functions. During plant growth, cells differentiate into various tissues and organs such as roots, stems, leaves, and flowers. Differentiation occurs in plants when cells from the root apical and shoot apical meristems, as well as the cambium, differentiate and mature to execute specialized roles. Living plant cells lose their ability to divide once they have been differentiated.  Plant Tissue Culture Totipotency Differentiation, Dedifferentiation and Redifferentiation Dedifferentiation: Plant cells that have already differentiated and lost the ability to divide regain the ability to divide and differentiate under particular conditions. Dedifferentiation is the term for this process. A dedifferentiated tissue has the potential to function as a meristem, giving rise to a new set of cells. The ability of those cells to differentiate further is influenced by a variety of factors, including genetic and epigenetic differences. This notion is utilized to create a callus in plant tissue culture.  Plant Tissue Culture Totipotency Differentiation, Dedifferentiation and Redifferentiation Redifferentiation: Redifferentiation is the process of dedifferentiated cells losing the ability to divide. The cells lose their ability to divide and differentiate once new cells are produced from the dedifferentiated tissues that act as meristems. They eventually mature in order to perform various roles within the plant body.  Plant Tissue Culture Totipotency Differentiation, Dedifferentiation and Redifferentiation Differentiation occurs in plants when cells from the root apical and shoot apical meristems, as well as the cambium, differentiate and mature to execute specialized roles. Living plant cells lose their ability to divide once they have been differentiated. However, under some circumstances, this power to divide further can be recovered. Dedifferentiation is the process by which mature cells reverse their differentiated state and acquire pluripotency. Redifferentiation is the process by which dedifferentiated cells lose their ability to divide and become specialized to perform a role by converting into a part of the permanent tissue. The main distinction between dedifferentiation and redifferentiation is this.  Plant Tissue Culture Totipotency Differentiation, Dedifferentiation and Redifferentiation Dedifferentiation is the process through which differentiated cells regain the ability to divide mitotically. As a result, the dedifferentiated tissue in the plant body functions as distinct meristematic tissue. As a result, this mechanism is critical for the generation of new cells at a specific site. Redifferentiation, on the other hand, is the loss of differentiated cells’ recovered ability to divide. It enables functional specialization of these cells, allowing them to execute a specific purpose inside the plant. The influence on the differentiated cells’ ability to divide is the major difference between dedifferentiation and redifferentiation. Plant Tissue Culture depends on: 1. Totipotency – ability of plant cells to regenerate into a whole 2. Plasticity – ability of the plant to alter their metabolism, growth and development to best suit their environment. Plant tissue culture are generally initiated from multicellular tissue fragments called explants which obtained from the living plants. Explants may originate form a wide range of plant tissue such as leaf, stem, root, petiole, hypocotyl, cotyledon, embryo or meristems. 1. The explant is selected (either haploid or diploid explant) - i.e. the part of a healthy plant which should be used as explants to produce haploid plant is pollen grain. Male part of the flower and it contains pollen grains. It is diploid in structure. 2. The plant growth can be achieved into two ways: Shoot directly by appropriate media By somatic embryogenesis - Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. Sterilization Sterilization methods used in Tissue culture Culture Laboratory – All the materials, e.g. Vessels, instruments medium, plant materials (used in culture work must be freed form microbes) Sterilization Techniques Sterilization is achieved by one of the following approaches: ▪ Dry heat treatment ▪ Flame sterilization ▪ Autoclaving ▪ Filter sterilization ▪ Wiping with 70% ethanol ▪ Surface Sterilization Culture Media Explants are then usually placed on the surface of a solid culture medium, but are sometimes placed directly into a liquid medium, when cell suspension cultures are desired. Culture media are generally composed of inorganic salts plus a few organic nutrients, vitamins and plant hormones.  As culture grow, pieces are typically sliced off and transferred to new media (sub cultured) to allow growth or to alter the morphology of the culture. Major Types of Media White’s medium – is one of the earliest plant tissue culture media MS medium – formulated by Murashige and Skoog (MS) is most widely used for many types of culture systems B5 medium – developed by gamborg for cell suspension and callus cultures and at present it modified form used for protoplast cultures N6 medium – formulated by Chu ad used for cereal anther culture Nitsch’s medium – developed by Nitsch and Nitsch and used for anther culture Steps in Plant Tissue Culture Techniques Types of Plant Tissue Culture Factors Affecting Tissue Culture Efficiency Plant regeneration from tissue culture varies with the following parameters: Plant species, Genotype within the species, Source of the culture tissue, Age and health of the donor plant, Nutrient medium, other factor Plant tissue Culture Applications The commercial production of plants used a potting, landscape, and florist subject To conserve rare or endangered plant species To screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance. Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant derived secondary metabolites and recombinant proteins used as biopharmaceuticals. Plant tissue Culture Applications To cross distantly related species by protoplast fusion and regeneration of the novel hybrid. To produced clean plant material from stock infected by viruses or other pathogens. Production of identical sterile hybrid species can be obtained Different in Plant tissue Culture and Animal Culture It grow on at specific temperature i.e normal temperature for human is 37% Carbon dioxide is also require It needed proper change in media otherwise cells will not grow properly Animal cells needed protein and hormones for proper development