Unit 1 Cell and Molecular Biology Section 3 Cell, tissue and plant tissue culture

Conditions required for bacterial and fungal growth An original stock of cells. The use of aseptic techniques when preparing media and cultures. A suitable container for cell growth. A growth medium providing essential nutrients.

Conditions required for bacterial and fungal growth (cont’d.) Opportunity for gas exchange – usually provided by bubbling air through the culture. Control of factors such as pH and temperature. A method of measuring cell growth – usually by monitoring optical density using a spectroscope.

Uses of fungal cultures Fungi such as yeast are used during :- Wine making Beer making Bread making Blue cheeses Antibiotics

Uses of bacterial cultures Bacteria are used in the production of yoghurt and cheese.

Conditions required for mammalian cell growth Mammalian cells are more fragile than microbial cells so require carefully controlled conditions. As well as the requirements given for microbial cells, mammalian cells also require a complex growth medium ( containing foetal bovine serum or FBS) as well as additives such as pH indicator, antibiotics and animal serum.

Production of mammalian cells Treating tissue with proteolytic enzymes (e.g. trypsin) to give a primary cell culture from which others can be obtained. These cloned cultures only have a limited life span as mammalian cells only divide a limited number of times.

Production of mammalian cells (cont’d.) Mammalian cells can also be obtained by using immortalised cell lines (e.g. hybridomas) derived from tumours. These cells are neoplastic and will produce tumours if transplanted. Mammalian cells require a solid surface for growth and grow as a monolayer until they cover the surface of the vessel. When the surface of the vessel is covered, the cells are said to be confluent/confivent and growth stops until a new culture with fresh media is created.

Uses for mammalian cells Mammalian cells are required for:- research into normal cell growth. production of new tissue for skin grafts replacement of important cells e.g. stem cells are being trialled to replace cells lost through Alzheimer's or to help regenerate nerves Production of antibodies for use in diagnostics and to help carry drugs to specific sites

Activity Read Dart Pg Scholar (Unit 1, ) Look at techniques in microbial culture and cloning cells for an interferon Bioassay

Plant Tissue Culture Plant tissue culture is used to create clones. This is useful To create pathogen free species Create crop species that have identical traits (e.g. height) Save endangered species Somatic Fusion (Protoplast hybridisation) is used to create new varieties. Plant tissue culture is then used to grow the plant

Somatic Fusion Cells from two different plants are selected Cell wall is removed using cellulase and pectinase Protoplasts are fused using either Polyethylene Glycol Electrical shock Protoplast is grown using tissue culture

Plant Tissue Culture Source of cell / tissue Small pieces of tissue are taken from a plant. These are called explants. Protoplast from somatic fusion. Plant cells / tissue have a long life span Suitable container Anything sterile (the entire procedure must be carried out under aseptic conditions).

Growth Medium Simple (can be liquid or solid) containing carbon source, minerals salts, water, vitamins Contains plant growth regulators to cause tissue differentiation e.g. Cytokinins promote shoot development Auxins promote root development

Events in Plant Tissue culture Explant is removed and sterilised. It is transferred to growth media A mass of undifferentiated cells called the callus forms (this occurs due to auxin and cytokinin concentrations being balanced). Tissue is transferred to growth media containing different concentrations of growth regulators to promote shoot and root production. Tissue culture plants acclimatised in greenhouses.

Totipotency Totipotency is the cell’s ability to differentiate. In theory, all cells have this potential as they have the same genome (nuclear totipotency) In practise, Plant cells are totipotent Mammalian cells vary e.g. Stem cells are totipotent

Activity Read Dart Pg Scholar (Unit 1, ) Create a table to compare the 3 types of tissue culture.