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Plant Cell Culture Reagents Photo by Harry F. Holloway, CCP and Jeff McAdams. Courtesy of Carolina Biological Supply Co.Tobacco callus serves as the starting material for cell culture. The culture of plant cells began as a method to simplify studies of plant biology, but applications of this technology have become big business. Many researchers are genetically engineering plants to be hardier or more nutritious. Others want to harness the plants' ability to produce beneficial compounds at will, especially since plant-derived products have been shown to combat human diseases such as malaria, heart disease, osteoporosis, and cancer. Of the many antitumor agents extracted from plants over the years, the most famous example comes from the bark of the Pacific yew tree. Paclitaxel was isolated in the early '70s, underwent clinical trials in the '80s, and was approved by the Food and Drug Administration (FDA) in 1992.1 Paclitaxel is the active ingredient in TAXOL®, the well-known cancer fighter marketed by Bristol-Myers Squibb of Princeton, N.J. TAXOL's use for the treatment of breast, ovarian, and non-small cell lung cancer and AIDS-related Kaposi's sarcoma elevated worldwide sales of the drug to $1.5 billion in 1999. Despite intense effort to synthesize this compound chemically, paclitaxel's complex structure can be fashioned only by members of the Taxus tree species. When extraction from bark became impractical, Bristol-Myers Squibb developed a semisynthetic production scheme that involved harvest of a related compound from the leaves of Himalayan yew trees. This intermediate could then be chemically modified to produce active paclitaxel. Although this provided a renewable source and did not kill the trees, the species grows only in Central Asia, creating international trade issues. The demand for paclitaxel's powerful antineoplastic properties has inspired both academic and industrial scientists to search for a way to culture Taxus cells and induce them to produce this valuable compound in vitro. In this effort, Bristol-Myers Squibb has teamed up with Phyton of Ithaca, N.Y., a company specializing in large-scale plant cell fermentation technology, and the process is awaiting FDA approval. Mitsui Petrochemical Industry of Japan has succeeded in commercially producing compounds by large-scale culture of plant cells.2 Among these products are the antibacterial agents berberine and shikonin and the nutraceutical ginseng. Interest in plant cell culture stems not only from its value in pharmaceutical production, but also its application to plant propagation and agriculture. Some use plant cell culture to simply propagate desirable plants, a field known as micropropagation. A large facet of plant biotechnology focuses on the genetic engineering of improved crops. The production of genetically modified foods often starts with the transfer of beneficial genes to plant cells in culture. This technology can produce crops that grow in poor soil or altered environments or have increased yields and resistance to disease, herbicides, or insects.3 Many are also using genetic engineering to increase the nutritional value of staples such as rice.4 "I believe there is a huge potential for improving the quality of edible crops," remarks Kenneth Torres, president of PhytoTechnology Labs of Shawnee Mission, Kan. Torres worked in the plant cell culture industry for 20 years before starting PhytoTechnology Labs, the only U.S. company focused strictly on products for the plant biotechnology market. Sigma's Phytatray is useful for regenerating whole plants from callus cultures. A Different Culture All of the applications mentioned above require reducing plants to suspended cell cultures and regenerating whole plants from these cultures. Initially establishing plant cell cultures may be the most challenging part of the process. The procedure starts with a sterilized explant cut from any part of a whole plant or a newly sprouted seed that is placed on semisolid media. The vessel is then sealed with Parafilm to prevent desiccation and incubated at 25°C in the dark.3 The plant responds to this injury by initiating rapid cell division around the site of the wound, and a mass of undifferentiated cells known as callus forms at the cut. Under the right conditions, callus formation usually occurs within one to five weeks. After passaging the callus material several times on solid media, a portion can be transferred to liquid media for suspended cultures. Callus liquid cultures are totipotent and retain the potential to synthesize compounds normally made by the whole plant. These cultures can also be manipulated to differentiate into organized tissue or whole plants. A crucial factor in establishing and maintaining callus cultures is the formulation of the media. Plant cell culture medium is complex. Sucrose (2 or 3 percent) typically serves as the carbon source. Numerous inorganic salts and a few organic supplements such as vitamins and amino acids are also required. The pH of plant media usually falls between 5.6 and 6.0. Since the most common media formulations have around 20 different components, many companies sell premade media, salt mixtures, or powders to add convenience and consistency to plant cell culture experiments. The most commonly used medium formulation was developed by T. Murashige and F. Skoog.5 This medium is commercially available in a wide variety of forms. Gamborg's B-5 medium is also commonly used and readily available.6 Sigma of St. Louis and PhytoTechnology Labs carry many other media formulations for specialized applications. The optimal medium formulation for a particular application depends on the plant species used and the application. The conditions for culturing common types of plant cells are well defined, but when working with a new species, growth conditions must be determined empirically. The medium formulation can also be altered to induce certain processes. Limiting the nitrogen or phosphate source can stimulate secondary metabolite production, and manipulating the carbon source can induce organogenesis or embryogenesis.7 PhytoTechnology Labs, JRH Biosciences of Lenexa, Kan., and ICN Biomedicals of Costa Mesa, Calif., can handle custom media requests. The source of gelling agent in solid growth media can also affect results with certain plant strains. When traditional agar is not tolerated, several agar substitutes such as Monsanto's Gelrite® Gellan Gum, Sigma's Phytagel™, and ICN Biomedical's Gel-Gro™ come in handy for making solid plant media. Both liquid and solid plant media support microbial growth, so good sterile technique is critical to prevent contamination. This is especially important because bacteria or fungi quickly dominate slow-growing plant cultures. PhytoTechnology Labs' distributes the Plant Preservative Mixture (PPM), a new product that inhibits the growth of bacteria and fungi in plant cell cultures. Growth of the Field Growth of plant cells in culture also requires addition of plant hormones called phytohormones or growth regulators. These messengers act at a distance like typical hormones but also function in the cell in which they're produced. Growth regulators profoundly affect plant cell growth, differentiation, and metabolism, and they can be exploited in culture to manipulate a host of activities including secondary metabolite production and regeneration.3 Most growth regulators used for plant culture can be classified as either auxins or cytokinins. The precise balance between auxins and cytokinins in the media is key to generating the desired effect. Auxins stimulate cell division in explants, and high auxin concentration suppresses organized growth and promotes formation of meristem-like cells. Use of the light-sensitive, natural auxin, indole-3-acetic acid (IAA), has been largely replaced by the more stable synthetic auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA). IAA, 2,4-D, and NAA are used for callus induction and growth, and IBA is used for regeneration. Auxinlike compounds such as Dicamba and Pichloram are also used for plant culture. Cytokinins promote cell division and regeneration, possibly through effects on tRNA and protein synthesis. Zeatin is a natural cytokinin that induces morphogenesis. Care should be taken not to autoclave solutions containing heat-sensitive zeatin. Commonly used synthetic cytokinins used for callus culture include kinetin, 6-benzylaminopurine (BAP), and N-isopentenylaminopurine (2iP). Undefined supplements such as coconut milk and fruit extracts can also be useful for callus and protoplast culture. Many companies sell growth regulators, with the widest selections available from Sigma, PhytoTechnology Labs, Life Technologies of Rockville, Md., and Carolina Biological Supply Co. of Burlington, N.C. Several companies make growth vessels especially for plant culture applications. Osmotek of Rehovot, Israel, manufactures innovative growth systems for plant tissue culture that are distributed by Life Technologies, Sigma, and Carolina Biological Supply. Gerald Tanny, founder and CEO of Osmotek, observes that many researchers are "developing unique [genetically modified] cultures but doing propagation with outmoded solid media technology because it's familiar to them." Osmotek's LifeRaft® vessels employ a membrane-based raft that floats on liquid media to promote improved exchange of nutrients compared to solid media systems. LifeRafts are ideal for regeneration of cell cultures into whole plants. Vessels made by Magenta Corp. of Chicago are also widely used and available through a number of distributors. PhytoTechnology Labs offers a new PhytoLabware™ culture tray and according to company representatives, Sigma's Phytatray™ and ICN's PlantCon™ vessels continue to be big sellers. Branching Out While serving plants well in support and defense, the rigid cell wall surrounding each plant cell can be an obstacle to plant biologists. This barrier prevents direct access to the cell membrane, precluding the use of antibodies or lectins to study membrane function.3 Cell wall components also contribute to the cell aggregation observed during plant cell culture, making single cell isolation difficult. For these reasons, some researchers prefer to study cells that have been stripped of their cell wall. These cells, surrounded only by a cell membrane, are known as protoplasts. Protoplasts will grow in culture, and a single protoplast can be induced to regenerate into a whole plant. Media for protoplast culture are similar to those used for normal plant cells, with the addition of an osmoticum (sorbitol or sucrose) to keep the medium isotonic. Agar can be toxic to protoplasts, so solid media should include agarose or other agar substitutes. One property often exploited by researchers is the ability of individual protoplasts to fuse, creating a hybrid organism. The biotechnological implications of this property continue to drive protoplast research. The future of plant biotechnology seems bright. Advances in the large-scale production of secondary metabolites from plant cell cultures are likely to drive this industry forward rapidly. Among the researchers developing plant cell culture systems to produce beneficial compounds is Jim Linden, professor of chemical and bioresource engineering at Colorado State University.8 Linden believes that "there will be some developments in fundamental metabolic engineering that will enhance a rate- limiting step in this process." Linden also foresees the"capability of using plant tissue culture to make transgenic plants that will produce chemicals in the whole plant, enabling standard agriculture as a production means," an approach termed "pharming." This proves what parents have been preaching for generations: "Eat your vegetables; they're good for you." And with recent advances in plant biotechnology, it looks like they're getting better every day. S Amy L. Francis can be contacted at afrancis@the-scientist.com. References 1. B.B. Rogers, "Taxol: a promising new drug of the '90s," Oncology Nursing Forum, 20:1483-9, 1993. 2. Y. Fujita, M. Tabata, "Secondary metabolites from plant cells--pharmaceutical applications and progress in commercial production," in Plant Tissue and Cell Culture, (C.E. Green et al., eds.), New York, Alan R. Liss Inc., 1987, pages 169-85. 3. A. Stafford, G. Warren (eds.), Plant Cell and Tissue Culture, Buckingham, U.K., Open University Press, 1991. 4. X. Ye et al., "Engineering the provitamin A (ß-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm," Science, 287:303-5, Jan. 14, 2000. 5. T. Murashige, F. Skoog, "A revised medium for rapid growth and bioassays with tobacco tissue cultures," Physiologia Plantarum, 15:473-97, 1962. 6. O. Gamborg et al., "Nutrient requirements of suspension cultures of soybean root cells," Experimental Cell Research, 50:151-8, 1968. 7. R.D. Hall, "An introduction to plant-cell culture," in Methods in Molecular Biology, Vol. 111:Plant Cell Culture Protocols, (R.D. Hall, ed.), Totowa, N.J., Humana Press, 1999, pages 1-18. 8. M. Phisalaphong, J.C. Linden, "Kinetic studies of paclitaxel by Taxus canadensis cultures in batch and semicontinuous with total cell recycle," Biotechnology Progress, 15:1072-7, November/December 1999.

Pharmers Market

May 1, 2000

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