YOUR GUIDE TO PLANT CELL CULTURE
(August 2003)
As the population expands there has been increasing concern over whether current rates of food productions can support the growing population. In response to the increasing demand, scientists and farmers have been working hard to come up with new ideas to meet these needs. These ideas range from simple procedures including selection of the best seeds to more complex procedures requiring the transplantation of genes into different plant species. This article focuses on one of the important aspects of plant science which is the growth of plant cells in an artificial media. This process is known as plant cell culture and can be applied in many different ways.
When carrying out a plant cell culture, there are three important things that must be considered. First the plant part of interest must be isolated from the intact plant. Next the appropriate environment to promote optimal growth must be discovered and applied. This may vary depending on the cells of interest. Finally, these procedures must be carried out in a sterile environment to prevent growth of microorganisms [1]. These topics are all discussed in addition to applications in which cell culture has been used.
Preparation
Growth conditions for plant culture must provide it with all essential nutrients which are normally provided by the plant itself. Furthermore, these conditions must be optimized to promote the growth of the particular cell type. The medium in which the cells are grown, must contain a carbon source, vitamins, salts and other organic supplements. The inorganic nutrients come in different concentrations of macronutrients and micronutrients. Macronutrients include nitrogen, potassium, calcium and magnesium, which are always found in a concentration greater than 0.5mM. The micronutrients include iron, copper, zinc, and cobalt and can be adjusted for maximum growth of each culture type. In addition, medium must provide vitamins which are usually synthesized by plants. In addition to vitamins and nutrients, growth hormones can also influence the growth rate of plants grown in artificial environments. Many plants grown in a conventional manner produce their own growth hormones, however, in culture, artificial hormones are supplied to ensure optimal growth of the plants.
Besides having the appropriate nutrients in the media, the maintenance of sterile conditions is essential for the success of the cell culture allowing it to be free of microorganisms. This requires that all equipment used in creating a cell culture must be sterilized to ensure contamination does not occur. There are many methods of ensuring sterilization with the use of alcohol, flame, and chemicals. Furthermore, containers should be covered at all times to ensure no further airborne contamination.
Culture types
Many plant species can be regenerated in vitro through several approaches but all require a starting point. This can be anything from a single cell that can be reproduced, a tissue or organ part, or a cut out piece of differentiated tissue (or organ) known as an explant. Once the starting point has been determined, the culture used to grow this part must be considered. There are many different plant cultures all useful for different things. Some of these culture types include the embryo culture, organ culture, callus culture, and cell culture. Figure 1 shows schematics of the different types of culture.
The main culture of interest is the cell culture, which allows for the culture of isolated cells from a very small cluster dispersed in a liquid media. This is the culture type that we will be focusing on throughout the remainder of this article.
Cell suspension cultures are rapidly dividing homogenous suspensions of cells grown in liquid nutrient media from which samples can be taken [2]. In a cell suspension, a mass of cells, called a callus, must first be collected. The callus can then be suspended in a liquid callus induction media containing all the required nutrients and elements to allow for optimal growth which acts to turn all cells into undifferentiated cells. The cell suspension is then placed on a shaker to allow the cell aggregates to disperse to form smaller clumps and single cells that are equally distributed throughout the liquid media. The cells will continuously grow until one of the factors becomes limiting causing cell growth to slow.
Before this happens, cells are collected and grown on a media containing hormones to activate specific cell growth. For example if the media contains auxin, it could activate formation of adventitious roots, whereas cytokinin would stimulate axillary and adventitious shoot proliferation and regulate differentiation. Once the plants have developed, they can be transplanted into soil where they can continue to grow.
Applications
Tissue culture techniques are becoming increasingly popular as an alternative means of plant vegetative propagation, mass production of chemicals, and genetic engineering [3]. The primary goal of plant tissue culture is crop management. This involves asexual methods of propagation to generate whole plants from single cells [1]. In order for successful selection and genetic manipulation to occur, one must have successful in vitro plant regeneration, making this topic very important.
In vitro clonal propagation is referred to as micropropagation [1]. In this context clonal means the production of genetically identical plants grown from parts of plants and reproduced by asexual reproduction. This procedure allows for many thousands of plants to be derived from a single cell or tissue in a relatively short amount of time [1]. This appears to be advantageous over conventional plant reproduction in that only a small amount of plant tissue is required as the initial explant for regeneration of millions of clonal plants in one year [1]. Therefore, this in vitro technique allows for speedy international exchange of plant materials. It also eliminates the danger of introducing disease to the crop if performed under sterile conditions. Furthermore, when crops are grown in vitro it allows for year round growth regardless of what Mother Nature throws at us. Unfortunately, it is very expensive to carry out clonal propagation due to the costly equipment and trained engineers to perform the procedures. In addition to high growth rates there could also be high contamination rates. Once contamination occurs it could spread through the crops causing great losses over a very short amount of time [1].
Cell cultures are also useful for the secondary metabolites they produce. Some of these metabolites that are a valuable source include flavors, natural sweeteners, industrial feedstocks, perfumes and commercial insecticides. These products do not perform vital physiological functions like amino acids or nucleic acids, but they are produced to ward off potential predators, attract pollinators, or combat infectious diseases [1]. Another useful metabolite produced by plants includes shikokin, which is a chemical used as both a dye and a pharmaceutical. Plant cell suspension culture is valuable for studying the biosynthesis of secondary metabolites. Although there are limitations of cell culture systems in producing secondary metabolites, they are favored over conventional cultivation methods. This is because of their ability to produce useful compounds under controlled conditions as well as their capability of using this technique to produce chemicals to meet market demands. In addition, specific cells of a plant can be multiplied to produce a higher yield of specific metabolites, which cannot be done through conventional methods of cultivation.
Disadvantages
As mentioned previously, contamination is one of the largest problems when dealing with cell cultures. Contamination becomes a problem because micro-organisms can grow much faster that plant cells and take up all the nutrients preventing the plants from growing. There are a variety of techniques to minimize the possibility of contamination. Autoclaving all media and equipment before use kills unwanted microbes. This involves heating to 1210C for 15-20 minutes at 20psi. Other methods to reduce risk of contamination include using a laminar flow hood with adequate air flow, keeping cell cultures in a room that is not commonly used, disinfect all fumehood surfaces that will be used. If contamination is still a problem, antibiotics are available with anti-mitotic components to reduce contamination for fungal and yeast problems.
Is Cell Culture good?
In response to growing demand of food to support an increasing population, biotechnology has allowed us to increase world agriculture allowing for a greater production of staple foods including wheat and rice. Using cell culture we can now produce a whole plant from a clump of cells artificially allowing crops to be grown through all seasons. In addition, methods have been introduced to permit a faster growing time and allow us to produce crops that can withstand severe conditions and insect infestations.
References
1. Chawla, H.S. 2000. Introduction to Plant Biotechnology. Science Publishers Inc. Enfield, NH, USA.
2. King, P.J. 1984. Induction and maintenance of cell suspension cultures. In: Cell Culture and Somatic Cell Genetics of Plants. (Vasil, I.K. ed) Vol. 1, pp 130-138. Academic Press, New York.
3. Pais, M.S., Mavituna, F., Novais, J.M. 1988. Plant Cell Biotechnology. Springer-Verlag Berlin Heidelberg, Germany.
(Art by Jiang Long – note that high res versions of these image files are available here)