Plant Cell Suspension Culture Lecture Notes PDF

Lecture 9 a Cell suspension Culture in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Understand the concept and significance of cell suspension culture. Learn the process of establishing and ma...

Lecture 9 a Cell suspension Culture in Plant Tissue Culture Prepared and presented by : Dr. Abdelaziz Mohamed Nasr Lesson Objectives Understand the concept and significance of cell suspension culture. Learn the process of establishing and maintaining cell suspensions. Discuss the applications and advantages of cell suspension culture. Cell suspension culture Unorganized plant cells can be grown as callus in aggregated tissue masses, or they can be freely dispersed in agitated liquid media. Techniques are similar to those used for the large-scale culture of bacteria. Cell or suspension cultures, as they are called, are usually started by placing an inoculum of friable callus in a liquid medium Cell suspension culture Under agitation, single cells break off and, by division, form cell chains and clumps which fracture again to give individual cells and other small cell groups. It is not always necessary to have a previous callus phase before initiating suspension cultures. Leaf sections floated on MS medium in the light showed rapid growth and cell division in the mesophyll, and after 4 days on a rotary shaker they can be disintegrated completely to release a great number of cells into suspension. Cell suspension culture Because the walls of plant cells have a natural tendency to adhere, it is not possible to obtain suspensions that consist only of dispersed single cells. Cultures of completely isolated cells have yet to be obtained. The proportion and size of small cell aggregates varies according to plant variety and the medium in which the culture is grown. As cells tend to divide more frequently in aggregates than in isolation, the size of cell clusters increases during the phase of rapid cell division. Cell suspension culture Because agitation causes single cells, and small groups of cells, to be detached, the size of cell clusters decreases in batch cultures as they approach a stationary growth phase. The degree of cell dispersion in suspension cultures is particularly influenced by the concentration of growth regulators in the culture medium. Auxinic growth regulators increase the specific activity of enzymes, which bring about the dissolution of the middle lamella of plant cell walls. Thus, by using a relatively high concentration of an auxin un differtiotiated and a low concentration of a cytokinin growth regulator in the culture medium, it is usually possible to increase cell dispersion. Cell suspension culture However, the use of high auxin levels to obtain maximum cell dispersion will ensure that the cultured cells remain undifferentiated. This may be a disadvantage if a suspension is being used to produce secondary metabolites. Cell suspension culture methods Batch cultures Cells are nurtured in a fixed volume of medium until growth ceases. Continuous cultures Cell growth is maintained by continuous replenishment of sterile nutrient media. Immobilised cell cultures Plant cells are immobilized in a variety of putatively inert matrices while fresh medium is continuously run. Cell suspension culture All techniques utilize some method of agitatting he culture medium to ensure necessary cell dispersion and an adequate gas exchange. Batch cultures Batch cultures are initiated by inoculating cells into a fixed volume of nutrient medium. As growth proceeds, the amount of cell material increases until nutrients in the medium are depleted or there is the accumulation of an inhibitory metabolite. Batch cultures have a number of disadvantages that restrict their suitability for extended studies of growth and metabolism, or for the industrial production of plant cells. They are widely used for many laboratory investigations. Small cultures are frequently agitated on orbital shakers onto which are fixed suitable containers, which range in volume from 100 ml (Erlenmeyer conical flasks) to 1000 ml (spherical flasks) Continuous cultures Plant cells are to be used for the large-scale production of a primary or secondary metabolite. Continuous culture techniques require fairly complicated apparatus. Agitation of larger cultures in bio-reactors is usually achieved by stirring with a turbine and/or by passing sterile air (or a controlled gaseous mixture) into the culture from below and releasing it through plugged vents. Mechanically stirred reactors damage plant cells by shearing. Notes on cell suspension culture The growth of plant cells is more rapid in suspension than in callus culture and is also more readily controlled because the culture medium can be easily amended or changed. Organs can be induced to develop in cell suspensions: root and shoot initiation usually commences in cell aggregates. Somatic embryos may arise from single cells. Cells from suspensions can also be plated onto solid media where single cells and/or cell aggregates grow into callus colonies from which plants can often be regenerated. Notes on cell suspension culture plants may be obtained from somatic embryos formed in suspensions. Once embryos have been produced, they are normally grown into plantlets on solid media. Cells from suspensions are plated onto solid media where single cells and/or cell aggregates grow into callus colonies from which plants can often be regenerated. Batch culture Continuous culture Immobilized culture Immobilized cell cultures This technique has only limited application to plant micropropagation, but is now employed quite widely when plant cells are grown for the production of their secondary products or for the bio-transformation of chemical compounds Assessment of cells in suspension culture Cell count Packed Cell Volume Cell Fresh Weight Fluorescein diacetate (FDA) Optical Density Viability test Fluorescein diacetate (FDA). Evans blue stains Evans blue stain What is the take-home message from today’s lecture. Let’s discuss. Any more questions?