Tissue CultureTissue CultureBiology

Plant Tissue CulturePlant Tissue Culture Lab - Atlanta Lab - Atlanta Botanical GardenBotanical Garden

oTissue culture, also known as micropropagation, is a propagation method used to produce plants under sterile conditions. oThis method uses plant explants (plant parts) or seeds that have been sterilized before being placed in containers with a growing medium (usually a gel) that has some nutrients added. oThe explants or seeds, the containers and the medium have all been sterilized, and this (if successful) prevents any cut or torn tissue, or the entire explant or seed itself, from becoming infected with a microorganism of some kind and rotting during the time these plant parts require to become rooted or to multiply.

oMany types of seeds germinated in these conditions tend to grow very fast compared to being sowed in standard growing media outside of sterile, enclosed containers. oUsing plant tissue, it is possible to grow exact copies of the donor plant. oThis is extremely useful for plants that genetically have desirable traits because one can create many clones of a particular plant much faster than traditional propagation methods like cuttings, pullings or divisions.The sterile nutrient media usually contains a nutrient solution (typically salts and vitamins), sucrose (sugar), hormones (optional), antibiotics (optional), and a solidifying or gelling agent such as agar (a product of seaweed).

Four stages of plant tissue culture have been defined by a scientific pioneer in the field, Toshio Murashige, professor emeritus of the University of California at Riverside:Stage I. Establishment of an aseptic (sterile) culture.Stage II. The multiplication of propagules (a propagule is any part of a plant used to make or become new plants).Stage III. Preparation of propagules for successful transfer to soil (rooting and "hardening" (acclimating) outside of sterile conditions in regular growing media).Stage IV. Establishment in soil (or other appropriate growing medium).

Tissue Culture Versus Tissue Culture Versus Traditional PropagationTraditional PropagationThe main difference between tissue culture and propagation done by cuttings, pullings, divisions or seeds is that the plants grow faster (the nutritious gel medium helps) and one can multiply them rapidly by giving them the right hormones and dividing them regularly. In theory, you can create an infinite number of plants from just one piece of tissue, and many plants in a relatively brief period of time. Below is a picture, courtesy of Jens Brettschneider, showing the difference in growth rate and plant size of seeds started at the same time, at left in tissue culture, and at right in traditional potting media.

Sterilizing explants (plant Sterilizing explants (plant tissue)tissue)Perhaps the most important part of Perhaps the most important part of having success with explants is having success with explants is selecting the right tissue and selecting the right tissue and starting with clean tissue. To starting with clean tissue. To dramatically increase your odds of dramatically increase your odds of success with an explant, keep the success with an explant, keep the plant that you plan to harvest the plant that you plan to harvest the explant from growing in very clean explant from growing in very clean conditions. You can even cover it conditions. You can even cover it with a plastic bag or grow it indoors with a plastic bag or grow it indoors under lights. Rain and wind carry all under lights. Rain and wind carry all kinds of bacteria and mold spores in kinds of bacteria and mold spores in them and onto the plant, making it them and onto the plant, making it very hard to get clean without very hard to get clean without killing the plant tissue. Select a killing the plant tissue. Select a piece of tissue that's clean, young piece of tissue that's clean, young and appears to be disease free.and appears to be disease free.

Plant Tissue Culture TechniquesPlant Tissue Culture Techniques

Plant tissue culture techniques are essential to many types of academic inquiry, as well as to many applied aspects of plant science. In the past, plant tissue culture techniques have been used in academic investigations of totipotency and the roles of hormones in cytodifferentiation and organogenesis. Currently, tissue-cultured plants that have been genetically engineered provide insight into plant molecular biology and gene regulation. Plant tissue culture techniques are also central to innovative areas of applied plant science, including plant biotechnology and agriculture.For example, select plants can be cloned and cultured as suspended cells from which plant products can be harvested. In addition, the management of genetically engineered cells to form transgenic whole plants requires tissue culture procedures; tissue culture methods are also required in the formation of somatic haploid embryos from which homozygous plants can be generated. Thus, issue culture techniques have been, and still are, prominent in academic and applied plant science.

Plant MaterialPlant MaterialPlants used in tissue culture need to be healthy Plants used in tissue culture need to be healthy and actively growing. Stressed plants, and actively growing. Stressed plants, particularly water-stressed plants, usually do particularly water-stressed plants, usually do not grow as tissue cultures. Insect and disease-not grow as tissue cultures. Insect and disease-freefreegreenhouse plants are rendered aseptic more greenhouse plants are rendered aseptic more readily, so contamination rate is lower when readily, so contamination rate is lower when these plants are used in tissue culture these plants are used in tissue culture procedures. Seeds that can be easily surface procedures. Seeds that can be easily surface sterilized usuallysterilized usuallyproduce contamination-free plants that can be produce contamination-free plants that can be grown under clean greenhouse conditions for grown under clean greenhouse conditions for later experimental use.later experimental use.

BackgroundBackgroundDuring seed formation, the developing embryo and During seed formation, the developing embryo and associated tissues tend to exclude pathogensassociated tissues tend to exclude pathogensand foreign materials that may be in the parent and foreign materials that may be in the parent plant. Contents of the seed, then, are essentiallyplant. Contents of the seed, then, are essentiallyaseptic and the resultant seedlings can be aseptic and the resultant seedlings can be maintained in the aseptic condition if the outer maintained in the aseptic condition if the outer surface ofsurface ofthe seed (seed coat) is sterilized with sodium the seed (seed coat) is sterilized with sodium hypochlorite (or other surface sterilant) prior tohypochlorite (or other surface sterilant) prior togerminating the seeds in a sterile petri dish.germinating the seeds in a sterile petri dish.

Plant hormones, like animal hormones, are relatively small molecules that are effective at low tissue concentrations. The two types of plant hormones used in this experiment are cytokinins and auxins.Cytokinins are derived from adenine and produce two immediate effects on undifferentiated cells: the stimulation of DNA synthesis and increased cell division (Ting, 1982). Cytokinins also produce a delayed response in undifferentiated tissue which is the formation of shoot primordia.Both naturally occurring cytokinins, such as zeatin and synthetic analogs, such as kinetin, demonstrate cytokinin effects (Figure 9.3). Although low tissue concentrations of cytokinins (e.g., 1 × 10-8 M zeatin) have noticeable effects, higher concentrations are found in actively dividing tissues such as those of plant embryos and developing fruits.

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