Research Tools for Plant Nucleic Acids

Plant Nucleic Acid Purification Kits Plant Nucleic Acid Purification Kits (continued) QIAGEN DNeasy Procedure Bio-Rad's portable Helios Gene Gun System By most accounts the world's population will increase to about 8 billion by 2025.1 Ironically, as population increases, arable land acres committed to agriculture are slowly but surely dwindling. Although major inroads, such as precision agriculture and resource conservation, are being made in farming technology, these new agricultural methods alone are not expected to produce crops at a rate to keep pace with the new and expanding global food demand. Biotechnology has come to the rescue with genetically modified organisms. Since the advent of molecular biology, scientists in the agricultural sector have been actively engaged in basic research to understand the molecular underpinnings of agriculturally and economically ("agronomically") valuable "traits" in plants. These qualities include increased starch, oil, and sugar production; resistance to herbivores and bacterial or viral infection; reduced nonproduct-yielding biomass such as leaves and stalks; increased tolerance to extreme environmental conditions such as alkalinity, drought, cold, and heat; and resistance to common agricultural herbicides. Researchers have also sought to develop mechanisms to introduce these new traits into plants and other organisms that exist in synergy with plant populations. These efforts have spawned an entire research industry dedicated to the development of novel herbicides, new growth enhancing fertilizers, and transgenic plants. Some of the most successful include the postemergence, broad-spectrum herbicide glyphosate (marketed as "Roundup(tm)" by Monsanto Co. of St. Louis), and subsequently a variety of "Roundup Ready(r)" (glyphosate-resistant) crops; Bacillus thuringiensis (Bt) protein (insect toxin)-producing transgenic cotton and corn, which resist insect predation; Papaya Ring Spot Virus (PRSV) coat protein-expressing transgenic papaya, which resists PRSV; and Flavr Savr(tm) tomatoes from Calgene of Davis, Calif. (now part of Monsanto), which resist postharvest handling damage. Plants present unusual technical hurdles and embody anatomical, physiological, biochemical, and genetic properties unparalleled in most other research systems. Protocols commonly used on mammalian or bacterial systems do not work with plants, and for those that do, the "borrowed" technique usually only works on one or very few similar plant types. Adding to these hurdles is the difficulty of introducing foreign genes into plants and plant cells, monitoring the expression of the engineered gene and/or gene product, and then producing and propagating a fertile, genetically engineered plant. Despite these many drawbacks, all is not bleak. Some of the more obvious advantages of working with plants are their diversity and relatively short life cycle as well as the ready supply of raw material and very little, if any, political and social opposition to research with plants. Recently the commercial availability of technologies and products optimized for plant biochemistry and genetic research--especially nucleic acid isolation, cloning, and gene expression analysis--has hastened plant research. This product profile will attempt to review a few of the commercially available tools that have and continue to be used successfully in plant research. Extraction, Purification, and Cloning of Plant Genomic DNA/RNA DNA and RNA isolation in plants is difficult. The structure and biochemistry of plants, particularly agronomically important ones such as soybean and corn, present unusual hurdles to successful extraction of the full complement of undegraded nucleic acid. The presence of high levels of natural phenolics, poly/ oligosaccharides, and other compounds that bind and/or copurify with DNA and RNA impede complete homogenization of tissue and extraction of nuclear material. Phenolic compounds in plants are notorious for their detrimental effects in both nucleic acid and protein research. They readily oxidize to form covalently linked quinones, which avidly bind nucleic acids, damaging and reducing the yield of nuclear material isolated. Generally, all nucleic acid extraction methods must be able to inactivate and/or remove the nucleic acid-damaging components from the homogenized cellular pool, while isolating nucleic acids from the potentially damaging components. QBIOGene Fast Prep kits represent one solution for plant nucleic acid isolation. Over the past three decades, hundreds of protocols for nucleic acid isolation from plants have been published. Many are variants of one procedure or another, optimized for a given plant species, organ, or cell type. Capitalizing on this wealth of accumulated technical knowledge as well as new materials and technologies, many providers of molecular biology products have developed kits specifically designed for plant nucleic acid purification that are applicable across a broad range of plant types. A list of these suppliers is provided in the accompanying table. QIAGEN of Valencia, Calif., has recently published a plant application guide titled "Plant Nucleic Acid Purification--Technical Hints and Applications," which includes information on topics such as sample collection, sample disruption, and typical applications. Once isolated, plant RNA and DNA may be used to construct a cDNA or genomic library, respectively, from any number of vectors,2 or contracted out to one of many custom library construction providers.3 Another option is to obtain a premade library available from a select few commercial entities that provide plant cDNA and genomic libraries, including InCyte Genomics (www.incyte. com), Life Technologies Inc., QBiogene (www.qbiogene.com), Research Genetics Inc. (www.resgen.com), and Stratagene (www.stratagene.com). Reporter Genes and Proteins Reporter or indicator genes encode a protein product that, when subjected to some physical or chemical treatment, can be "visualized" and thereby serve as an indicator of gene expression. These proteins have become important tools in plant genetic engineering in that they facilitate visual assessment of successful transformations without extensive characterization of all prospective transformants. Although some reporter genes may serve as selectable markers (those conferring antibiotic or herbicide resistance, for example), very few selectable markers are true reporter genes. Some of the earliest reporter genes used in plant genetic engineering were the cat and uidA (or gus) genes, which encode the chloramphenicol acetyltransferase and ß-glucuronidase proteins, respectively. When transformed cells expressing the gus gene are treated with an appropriate enzymatic substrate, they impart a vivid color. This easily allows the visual identification of individual cells or regions of tissue--e.g., a plant leaf or fruit--that contain expressed reporter protein.4 The gus reporter has served very well as a marker of gene expression during plant development and as an indicator of successful Agrobacterium tumefaciens-mediated transformation. It has also contributed to much of our modern knowledge of promoter function in plant and animal cells. Another reporter gene, lux, isolated from the North American firefly, Photinus pyralis, came into widespread use in the late 1980s and early 1990s. Another version of this reporter gene was recently isolated from the bioluminescent bacterium, Vibrio harveyi. The lux gene product, luciferase, emits light when incubated with adenosine triphosphate and the substrate, D-luciferin. This light can be measured with a luminometer, by scintillation spectroscopy or by photography. New technologies are advancing plant research. Unfortunately, each of these reporter gene technologies destroys the biological material being assayed, representing a major hurdle to continued plant genetic engineering progress. What was needed was a reporter system that could be monitored with very little, if any, destruction or perturbation of the biological material. A new generation of fluorescent reporter genes, made possible by the observation that certain organisms contain proteins that fluoresce under certain light conditions, helped scientists override this hurdle. Subsequently, researchers isolated the genes to these "reporter" proteins and included them in cloning cassettes.5 Some of the more commonly used fluorescent protein genes are the jellyfish (Aequorea victoria) and sea pansy (Renilla reniformis) green fluorescent proteins (GFPs), and the IndoPacific sea anemone (Discosoma sp.) red fluorescent protein (RFP). By genetically modifying the amino acid composition of GFP, researchers have been able to change its' light absorption/emission characteristics, creating yellow (YFP) and blue (BFP) fluorescent proteins, respectively. Using these new reporter constructs, successfully transformed cells--those expressing the reporter gene protein product--could easily be detected by subjecting the cells or plant tissue to a specific wavelength of light and observing the emitted light, or fluorescence. Researchers now had a noninvasive mechanism to determine if the desired cloned gene was being expressed. The added benefit of this technology was that the reporters could be utilized in novel ways. For example, fluorescent reporter-promoter fusions have been used to investigate promoter function in vivo. These analyses also have been extended to monitor promoter and gene function in plants during development and under various environmental stresses. The detailed cellular analyses afforded by the fluorescent reporter, or indicator, proteins have also provided researchers with unique opportunities to assess the spatial and temporal distribution of proteins in living cells. Additionally, they also permit researchers to study protein-protein interactions as well as interactions between proteins and small molecules that occur during cell signaling and enzyme activation and inactivation. Unfortunately there are no plant- specific fluorescent reporter gene vectors/cassettes available. But many commercial suppliers provide a broad variety of vectors containing reporter genes, from which the creative researcher can construct a reporter cloning vector/cassette customized to their plant research needs. Many of these suppliers also provide substrates, reagents, and packaged assay kits for many of the reporter systems described above. Further information on these technologies, both basic and detailed, can be found on the CLONTECH, Molecular Probes, QBiogene, Promega, and Stratagene Web sites or by referring to "Plant Gene Transfer and Expression Protocols."6 Gene Transfer There are essentially three methods by which DNA ("foreign genes") can be introduced into plant cells or tissue: direct DNA uptake into protoplasts, A. tumefaciens Ti vector-mediated transfection, and biolistic particle bombardment of cultured plant cells and tissue. Direct DNA uptake into protoplasts is in many ways analogous to plasmid transformation of "competent" bacterial cells, which are specially treated--or made "competent"--to optimize naked DNA absorption. Plant cells can be treated to remove the cell wall--producing a "protoplast"--and to neutralize the electrostatics of the naked cell membrane, facilitating the uptake of a plasmid construct into the plant cell. The preparation of protoplasts requires a cocktail of cell wall digesting cellulases and pectinases in an isoosmotic solution. Once transformed, protoplasts can be induced to regenerate cell wall and callus and, with the right combination of the plant hormones auxins and cytokinins, whole plants as well. A. tumefaciens Ti vector-mediated transfection (AT) technology is the product of the observation that in tissue infected with crown gall disease, the tumor-inducing Ti plasmid within A. tumefaciens can stably insert part of its DNA into the host genome, where it is transcribed and passed through seed to progeny. Research scientists have capitalized on this observation, removing parts of the Ti plasmid and introducing new elements that allow the introduction of foreign DNA into the plasmid. The new Ti construct maintains its ability to shuttle the foreign genes into the host genome, so when specially conditioned tissue or protoplasts are subjected to infection by engineered Ti plasmid-containing A. tumefaciens, the desired foreign genes can be introduced into plant cells. Unfortunately, this process has largely been successful only with dicotyledonous plants such as tobacco and petunia. Recent reports over the past few years have demonstrated successful AT-mediated transformation of some monocotyledonous species,7 so there is hope that this technology may come to play a major role in plant genetic engineering. Most Ti cloning vectors and A. tumefaciens hosts are obtained from noncommercial sources such as colleagues involved in genetic engineering at other research institutions, or from germ stock banks, such as the American Type Culture Collection of Manassas, Va. Biolistic Particle Delivery: the "Gene Gun" Some of the major limitations to genetic engineering of highly desirable plants are the extreme difficulty of isolating and maintaining viable protoplasts, the inefficiency of transfection using protoplast direct DNA update and AT-mediated transformation, and in particular, the inability to regenerate complete--much less fertile--plants from transformed cells. Researchers have found an ingenious solution to many of these problems. Why not just shoot the DNA into plant tissue? This idea revolutionized not only how researchers could manipulate the genomes of plants, but also their study of fundamental cellular processes. The process, "biolistic" (biological and ballistic) particle delivery, was the invention of plant researchers at the New York State Agricultural Experiment Station in Geneva, N.Y., and the Cornell Nanofabrication Facility at Cornell University in Ithaca, N.Y., in the early 1980s. The technology patent (U.S. Patent Office No. 4,945,050), titled "Method for transporting substances into living cells and tissues and apparatus therefor," describes a device, a gun, and a procedure for literally shooting inert or biologically active particles such as DNA into tissue or cells. The inventors and fellow researchers first reported its use in genetic transformation of maize9 and tobacco10 in two keynote papers in 1988. Reviews on this technology are also available.11,12 This technique provided for the first time a highly effective means by which researchers could genetically transform monocotyledons--the agronomically important cereal plants such as corn, wheat, sugarcane, and barley. The "gene gun" was a mechanism for introducing novel genes into intact tissue, cultured plant cells, and subcellular organelles (mitochondria and chloroplasts). One of the major successes of this technology was the introduction of certain PRSV genes into papaya, which induced resistance to the dreaded virus that was killing the Hawaiian papaya industry in the early 1990s. The gene gun technology has since been very useful in genetically modifying other target crops and introducing "reporter genes" into cells and plants to study gene and protein expression, photosynthesis, and other physiological and biochemical cellular processes. The gene gun uses an inert gas to accelerate microscopic tungsten or gold beads coated with DNA molecules toward the target cells or intact tissue. Like microscopic bullets, the particles tear through the seemingly impervious cell wall and membrane and deliver the genetic material into the cell. Although it is quite literally a shot-gun approach--the cells receiving the DNA and the location within the cell where the DNA ends up are indeterminate--by carefully screening transformed cells, researchers can usually isolate one or several "transformed" cell lines bearing the genotype and phenotypic traits they were attempting to engineer into the cells. During the early years after this technology was introduced, many "home grown," in-house variants of a biolistic particle delivery system were built, using air-charged BB and pellet guns and .22 caliber gunpowder charges, but most were absolute failures. The grandson of the original "Cornell" device and the current commercially available biolistic particle delivery device is the Biolistic PDS-1000/He System from Bio-Rad Laboratories Inc. of Hercules, Calif. This device has been utilized successfully in many projects, as evidenced by the plethora of research publications in journals (including The Plant Cell, Plant Molecular Biology, Plant Cell Reports, and The Plant Journal) that cite the use of the Biolistic PDS-1000. A portable (hand-held) version of a biolistic delivery device, the Helios Gene Gun System, is also available from Bio-Rad Laboratories. The repertoire of research tools specifically designed and/or customized for molecular plant research is small in comparison to their mammalian and bacterial counterparts, but it is slowly and surely growing. The growing demand for increased food production to feed the masses in the 21st century, and the renewed interest in plants as invaluable sources of pharmacological compounds, are driving research efforts to develop novel technologies and tools specifically designed to aid and enhance plant molecular research. The plant researcher is encouraged to exploit these new tools to further their research. To keep abreast of these new tools, LabConsumer will continue to keep its readers on the cutting edge of life science advances. Christopher M. Smith can be contacted at csmith@sdsc.edu. References 1. Division of Agriculture and Natural Resources, University of California, "Future in focus: 2000-2025 - Agriculture," California Agriculture, July-August, 2000. 2. L. DeFrancesco, "Don't clone alone: a profile of cDNA libraries and kits," The Scientist, 11[18]:16, Sept. 15, 1997. 3. B. Johnson, "We deliver: a profile of molecular biology services," The Scientist, 13[21]:20, Oct. 25, 1999. 4. D. Wilkinson, "Gene analyses--sunny side up!" The Scientist, 13[7]:18, March 29, 1999. 5. M. Chalfie et al., "Green fluorescent protein as a marker for gene expression," Science, 263:802-5, 1994. 6. H. Jones, ed., Plant Gene Transfer and Expression Protocols. Methods in Molecular Biology Series, No. 49, Humana Press, Totowa, N.J., 1995. 7. G. De la Riva et al., "Agrobacterium tumefaciens: a natural tool for plant transformation," Electronic Journal of Biotechnology, 1(3), December 15, 1998. 8. J. J. Lin et al., "Effects of Agrobacterium cell concentration on the transformation efficiency of tobacco and Arabidopsis thaliana," Focus (Life Technologies) 16(3):72-7, 1994. 9. T. Klein et al. "Transfer of foreign genes into intact maize cells with high-velocity microprojectiles," Proceedings of the National Academy of Sciences, 85:4305-9, 1988. 10. T. Klein et al., "Stable genetic transformation of intact Nicotiana cells by the particle bombardment process," Proceedings of the National Academy of Sciences, 85:8502-5, 1988. 11. T. Klein et al., "Transformation of microbes, plants and animals by particle bombardment," Bio/Technology, 10:286-91, 1992. 12. I. Potrykus, "Gene Transfer to plants: assessment of published approaches and results," Annual Review of Plant Physiology and Plant Molecular Biology, 42:205-25.

Research Tools for Plant Nucleic Acids

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