Following the initial wash, explants are then transferred to a Clorox ® solution (generally 10 percent Clorox which makes a solution with 0.525 percent sodium hypochlorite - the active ingredient), or to an alcohol solution (usually 70 percent ethanol). The explant is submerged in the Clorox for various times, but 10 minutes often works well for many plants. If you use ethanol, the time is generally shorter, with many tissues ready after one minute of exposure.
When the explant has been surface sterilized, it is usually removed from the sterilizing solution and rinsed several times in sterile, distilled water. This last step is performed inside a laminar flow hood to maintain the axenic condition of the explant and to prevent the re-introduction of contaminating microbes. The explant is then ready to be trimmed if necessary and placed onto a tissue culture medium.
There are many different formulations of media, and the one selected will depend largely upon the plant species and the cell, tissue or organ selected for culture. One of the most widely employed media is the medium developed by Toshio Murashige and Folke Skoog (Murashige, T. and F. Skoog. 1962. Physiologia Plantarum 15:473-497). The Murashige and Skoog medium is so well-known to the scientific community that it is often abbreviated simply as the MS medium. Other important media formulations include Gamborg's B-5 medium and McCown's Woody Plant medium. There are others too numerous to mention that are used for specific purposes.
Basically a nutritive medium contains macronutrients (minerals required in rather large concentrations such as N, P, K, Mg, S, Ca), micronutrients (also called trace elements because they are required in low concentrations such as Fe, Zn, B, I, Mn, Mo, Co, Cu), vitamins (thiamine, nicotinic acid, riboflavin most commonly), sugars (sucrose and glucose are common) and sugar alcohols (myo-inositol in particular). A medium may also include amino acids, nucleic acid bases, or other organic molecules. If the medium has no phytohormones or plant growth regulators, it is referred to as a basal medium.
Phytohormones or plant growth regulators may be added to the basal medium to stimulate the growth and development of the explant in a particular fashion. There are five classes of phytohormones: auxins, cytokinins, gibberellins, abscisic acid and ethylene. Auxins and cytokinins are most commonly employed in tissue culture media.
In general, auxins are promotive of root initiation, although they may inhibit root elongation and subsequent development. In general, cytokinins tend to promote the development of new vegetative buds or the opening and growth of existing vegetative buds. There are many exceptions to those generalizations, as is often the case with generalizations.
Folke Skoog and Carlos Miller, in a classic study, demonstrated the interaction of auxins and cytokinins when growing tobacco pith cells (Skoog, F. and C.O. Miller. 1957. Symp. Soc. Exp. Biol. 11:118-130). They observed that when tobacco pith was exposed to a medium containing a high auxin concentration relative to the cytokinin concentration that roots formed from the pith tissue. They also observed that when pith was exposed to a medium containing a high cytokinin concentration relative to the auxin concentration that shoots developed from the pith tissue. When the concentrations of the growth regulators were relatively balanced, no organs were formed, but instead the pith cells proliferated in an unorganized fashion to form a tissue called callus.
Once a medium formulation has been selected and the solution is prepared, the pH of the medium must be adjusted. Many plant culture media are designed for a pH range from 5.7 to 5.8. This pH range keeps most of the ions in a charge state where they are available for absorption by the plant cells.
Once a medium contains the proper concentration of nutrients, is at the proper pH, and the agar is dissolved (if you are using agar), the medium is dispensed into a culture vessel. This may a test tube, a baby food jar, a canning jar, or a specialized culture vessel such as a Magenta Vessel ( TM Magenta Corp.). The container is capped with either a gas-permeable lid or a lid that vents to the atmosphere without letting microbes inside easily. The medium then is sterilized either by heating under pressure in an autoclave (generally 15 min at 15 psi, 121 C). Alternatively, the medium may be sterilized by filter-sterilization through a membrane that will filter out microbes (45 micrometer pore size or less) and then dispensed into sterile culture vessels.
Smith, Roberta H. 1992. Plant Tissue Culture: Techniques and Experiments. Academic Press, Inc. N.Y. (ISBN 0-12-650340-0)
[An excellent source of experiments aimed toward the undergraduate student population, although high school to graduate students would find it useful, with background information, technical tips and media formulations. Suppliers are also listed]
Dodds, J. H. and L. W. Roberts. 1995. Experiments in plant tissue culture. 3rd ed. Cambridge University Press. N.Y. (ISBN 0-521-47892-8)
[Aimed at the college and professional audience, this may be of less interest to the novice or generalist, but is certainly useful for individuals wanting more specialized techniques. Detailed instructions to carry out the procedures are provided along with suppliers.]
In Vitro Cellular and Developmental Biology - Plant
Published by the Society for In Vitro Biology
Plant Cell Reports
Plant Cell, Tissue and Organ Culture
Carolina Biological Supply Company
2700 York Road
Burlington, NC USA 27215
(Chemicals, prepared media, glassware, and live cultures are all carried by Carolina Biological.)
Sigma Chemical Company
P.O. Box 14508
St. Louis, MO USA 633178
(A good source of fine chemicals. Ask for the tissue culture department for specific requests.)
3175 Staley Road
Grand Island, NY USA 14072
(Carries an extensive line of prepared media, also has chemicals and some labware available.)
P.O. Box 13481
Shawnee Mission, KS USA 66282
(Carries prepared media, media components, and supplies. Will do custom media formulations.)
Bellco Glass, Inc.
340 Edrudo Road
P.O. Box B
Vineland, NJ USA 08360
(A source of culture vessels including specialized flasks and tubes. Also carries lab equipment.)
Much of the glassware and equipment, and some culture media may be obtained from other general laboratory suppliers such as Fisher Scientific or VWR Scientific.