A HISTORY OF PLANT BIOTECHNOLOGY : FROM THE CELL THEORY

The foundations of modern plant biotechnology can be traced back to the Cell Theory of Schleiden (Arch Anat Physiol Wiss Med (J Müller) 1838:137–176, 1838) and Schwann (Mikroscopische Untersuchungen über die Übereinstimmung in der Struktur und dem Wachstum des Tiere und Pflanzen. W Engelmann: Leipzig No 176, 1839), which recognized the cell as the primary unit of all living organisms. The concept of cellular totipotency, which was inherent in the Cell Theory and forms the basis of plant biotechnology, was further elaborated by Haberlandt (Sitzungsber K Preuss Akad Wiss Wien, Math-Naturwiss 111:69–92, 1902), who predicted the production of somatic embryos from vegetative cells. This brief historical account traces the development of technologies for the culture, regeneration and transformation of plants that led to the production of transgenic crops which have become central to the many applications of plant biotechnology, and celebrates the pioneering men and women whose trend-setting contributions made it all possible.
Keywords Cell theory - Genetic transformation - Plant regeneration - Totipotency - Transgenic crops
Opening Plenary Address delivered at the international conference on “Plants for Human Health in the Post-Genome Era”, held August 26–29, 2007, in Helsinki, Finland.
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Plant biotechnology is founded on the demonstrated totipotency of plant cells, combined with the delivery, stable integration, and expression of transgenes in plant cells, the regeneration of transformed plants, and the Mendelian transmission of transgenes to the progeny. The concept of totipotency itself is inherent in the Cell Theory of Schleiden (1838) and Schwann (1839), which forms the basis of modern biology by recognizing the cell as the primary unit of all living organisms. The Cell Theory received much impetus from the famous aphorism of Virchow (1858), “Omnis cellula a cellula” (All cells arise from cells), and by the very prescient observation of Vöchting (1878) that the whole plant body can be built up from ever so small fragments of plant organs. However, no sustained attempts were made to test the validity of these observations until the beginning of the 20th century because the required technologies did not exist and the nutritional requirements of cultured cells were not fully understood (see Gautheret 1985). Haberlandt (1902) was the first to conduct experiments designed to demonstrate totipotency of plant cells by culturing isolated leaf cells in diluted Knop’s (1865) nutrient solution. He failed largely because of the poor choice of experimental materials (even now, more than 100 years later, there are only rare instances where intact leaf cells have been cultured successfully), inadequate nutrients, and infection (see Vasil and Vasil 1972). Nevertheless, he boldly predicted that it should be possible to generate artificial embryos (somatic embryos) from vegetative cells, which encouraged subsequent attempts to regenerate whole plants from cultured cells.
The following pages provide a short history of the evolution of a variety of ideas and technologies that are now routinely used for the genetic improvement of plants, and celebrate the many pioneering men and women who played key roles in the development of plant biotechnology. It does not include the history of plant tissue culture, which can be found elsewhere (White 1943; Gautheret 1985), and the use of plant cell cultures or transgenic plants for the production of pharmaceuticals, vaccines, etc., as these subjects are covered adequately elsewhere in this volume.
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Development of nutrient media
Initial progress in the culture of plant tissues came from the work of Molliard (1921) in France, Kotte (1922) in Germany, and Robbins (1922) in the United States, who successfully cultured fragments of embryos and excised roots for brief periods of time. The development of improved nutrient solutions, informed choice of plant material, and appreciation of the importance of aseptic cultures, led to long-term or indefinite cultures of excised tomato roots, and cambial tissues of tobacco and carrot, by White (1934, 1939) in the United States, and Gautheret (1934, 1939) and Nobécourt (1939) in France. The discovery of the naturally occurring auxin indole-3-aetic acid (IAA) and its beneficial effects on plant growth (Went 1928; Kögl et al. 1934; Thimann 1935), soon led to its incorporation in plant nutrient media (see White 1943; Gautheret 1985).
White (1943) and others believed that the nutrient solutions based on Knop’s (1865) and other formulations neither provided optimal growth nor were stable or satisfactory over a wide range of pH values. These concerns led to the development of White’s (1943) medium, which was widely used until the mid-1960s. During this period a systematic study of mineral and other requirements of plant tissues grown in culture was carried out (Hildebrandt et al. 1946; Heller 1953), demonstrating the need for a greatly increased level of mineral salts in the medium (see Ozias-Akins and Vasil 1985). In a similar study, designed to optimize the growth of cultured tobacco pith tissue, a marked increase in growth obtained by the addition of aqueous extracts or ash of tobacco leaves to White’s medium was found to be caused largely by the inorganic constituents of the extracts, leading to the development of the first chemically defined and most widely used nutrient solution for plant tissue cultures (Murashige and Skoog 1962). The principal novel features of the new medium were the very high levels of inorganic constituents, chelated iron in order to make it more stable and available during the life of cultures, and a mixture of four vitamins and myo-inositol.
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Discovery of cytokinins and their role in morphogenesis
The initial success in obtaining unlimited growth of plant tissues was limited to the use of explants that contained meristematic cells. Continued cell division and bud formation were soon obtained when tobacco pith tissues that contained mature and differentiated cells were cultured on nutrient media containing adenine and high levels of phosphate (Skoog and Tsui 1951). However, cell divisions occurred only when the explant included vascular tissue (Jablonski and Skoog 1954). A variety of plant extracts, including coconut milk, (the beneficial effect of coconut milk, the liquid endosperm of immature coconuts, on plant tissue cultures was first shown by van Overbeek et al. 1941) were added to the nutrient medium in an attempt to replace vascular tissues and to identify the factors responsible for their beneficial effect. Among these, yeast extract was found to be most effective and its active component was shown to have purine-like properties. This finding led to the addition of DNA to the medium which greatly enhanced cell division activity (see also Vasil 1959). These investigations resulted in the isolation of kinetin from old samples of herring sperm DNA (Miller et al. 1955), and the understanding of the hormonal (auxin-cytokinin) regulation of shoot morphogenesis in plants (Skoog and Miller 1957). Later studies led to the isolation of naturally occurring as well as many synthetic cytokinins, the elucidation of their role in cell division and bud development, and their extensive use in the micropropagation industry related to their suppression of apical dominance resulting in the development of many axillary shoots.
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Large-scale culture of plant cells
Mass culture of plant cells in suspension culture to produce natural products was first proposed by Routier and Nickell (1956), and Tulecke and Nickell (1959). Although it is now possible to economically produce a few products such as shikonin, catharanthine, ginseng, paclitaxel, etc., in continuous cultures in 50,000L or larger bioreactors, further exploitation of this technology has been limited owing to continuing technical and biological problems, and high costs (see Staba 1980; Constabel and Vasil 1987, 1988). Consequently, attention has shifted during the past 25 years to using transgenic plants, rather than cell cultures, as ‘biofactories’ for the production of a variety of pharmaceuticals, vaccines, etc. (these are described in detail in other chapters in this volume; see also Ma et al 2005; Fox 2006; Murphy 2007).
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Somatic embryogenesis
The establishment of efficient embryogenic cultures has become an integral part of plant biotechnology as regeneration of transgenic plants in most of the important crops—canola, cassava, cereals, cotton, soybean, woody tree species, etc.—is dependent on the formation of somatic embryos. One of the most attractive features of embryogenic cultures is that plants derived from them are predominantly normal and devoid of any phenotypic or genotypic variation, possibly because they are derived from single cells and there is stringent selection during embryogenesis in favor of normal cells (see Vasil 1999). Embryogenic cultures were first described in callus and suspension cultures of carrot, grown on coconut milk-containing media, by Reinert (1958) and Steward et al. (1958), respectively. With increasing understanding of the physiological and genetic regulation of zygotic as well as somatic embryogenesis, embryogenic cultures can now be obtained on chemically defined media in a wide variety of species (Thorpe 1995; Raghavan 1997; Vasil 1999; Braybrook et al. 2006). In most instances the herbicidal synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) is required for the initiation of embryogenic cultures; somatic embryos develop when such cultures are transferred to media containing very low amounts of 2,4-D or no 2,4-D at all.
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Androgenic haploids
Haploids are a valuable commodity for plant breeders but they are seldom used as they occur only rarely in nature. This problem was greatly resolved by the experimental induction of haploidy in cultured anthers of Datura innoxia (Guha and Maheshwari 1966), which like many great advances in science was a chance discovery (Guha-Mukherjee 1999). The technology was further defined and improved by the work of Nitsch and Nitsch (1969). It is now well established that it is possible to shift the developmental pattern of microspores from a gametophytic to a sporophytic phase simply by excising and culturing the anthers at the microspore stage of development. The microspores, instead of forming pollen grains, give rise to somatic embryos either directly or after producing a callus. The resulting plants are either haploids or homozygous diploids (dihaploids) as a result of spontaneous or experimentally induced diploidization. Although the impact of androgenic dihaploids has been limited, they have been used to identify breeding lines with desirable traits and for the production of improved varieties of tobacco, wheat, rice and other crops.
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Totipotency of plant cells
During the 1950s a number of attempts were made to demonstrate the totipotency of plant cells. The first evidence of the possibility that single cells of higher plants could be cultured in isolation was provided by Muir et al. (1954), who obtained sustained cell divisions in single cells of tobacco placed on a small square of filter paper resting on an actively growing callus, which served as a nurse tissue. Similar results were obtained by Bergmann (1959) who plated single cells and cell groups suspended in an agar medium. Further progress was made by Jones et al. (1960), who were able to culture single isolated cells in a conditioned medium in specially designed microculture chambers. Each of these studies highlighted the importance of the nurse tissue or the conditioned medium for the survival and growth of the isolated cells. Direct and unequivocal evidence of the totipotency of plant cells was finally provided by Vasil and Hildebrandt (1965, 1967), who regenerated flowering plants of tobacco from isolated single cells cultured in microchambers, without the aid of nurse cells or conditioned media. This fulfillment of Haberlandt’s (1902) prophecy, combined with the more recent success in the genetic transformation of plants (see below), continues to provide the theoretical and conceptual basis of plant biotechnology.
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Protoplasts and somatic hybridization
In a series of studies during the early 1960s, Cocking (1960, 2000; see Vasil 1976; Giles 1983) described methods for the isolation of plant protoplasts by incubating segments of roots and other tissues in crude mixtures of cell wall hydrolyzing enzymes. The small number of protoplasts that were isolated were used mostly for physiological studies. Two key developments in 1970 and 1971 pointed to the potential use of protoplasts for plant improvement: (i) Induced fusion of protoplasts of diverse species (Power et al. 1970) as a means to produce somatic hybrid cells and novel hybrid plants without regard to taxonomic relationships. (ii) The regeneration of plants from cultured protoplasts (Takebe et al. 1971). Plants can now be regenerated from protoplasts of a wide range of species. Similarly, a variety of somatic hybrids have been obtained between related as well as unrelated species, although useful hybrids have been produced only in a limited number of species, such as Brassica, Citrus and Solanum. Protoplasts have, however, proven to be very useful in genetic transformation of plants (Marton et al. 1979; Davey et al. 1980; Paszkowski et al. 1984), including the economically important cereals (Vasil and Vasil 1992).
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Genetic transformation and transgenic crops
Even after extensive world-wide efforts, embryo-rescue, androgenetic haploids, somatic hybrids, and the much touted somaclonal variation (Larkin and Scowcroft 1981), have not lived up to their purported promise in becoming important and useful means of creating novel genetic variability for crop improvement. Indeed, much of the success during the past quarter century in the production of plants with novel and useful traits has been based on the infinitely more precise and predictable methods of genetic transformation, in which well characterized single or multiple genes introduced into single cells become stably integrated into the nuclear genome and are transmitted to progeny like dominant Mendelian genes.
Three principal methods have been developed for the introduction of genes into plants: (i) Agrobacterium tumefaciens-mediated gene transfer. (ii) Direct DNA delivery into protoplasts by osmotic or electric shock. (iii) Direct DNA delivery into intact cells or tissues by high velocity bombardment of DNA-coated microprojectiles (the biolistics procedure).
It is well known that the crown-gall disease of plants is caused by the soil-borne bacterium Agrobacterium tumefaciens. An early study by Braun (1958) demonstrated the transformed nature of tumor cells as they could be freed of the bacteria and still grown indefinitely in hormone-free media; non-transformed cells needed media supplemented with an auxin and a cytokinin for continued cell divisions. He proposed that a Tumor Inducing Principle (TIP) present in the bacteria was responsible for transformation. Bacteria-free tumor cells were found to contain large amounts of opines, a new class of metabolites (Petit et al. 1970). The critical observation that the presence of a particular opine—octopine or nopaline—in the bacteria-free tumor cells was determined by the bacterial strain used for tumor formation, rather than the plant, suggested that a functional bacterial gene had become a part of the plant cell. This was a revolutionary idea that was initially met with considerable skepticism.
These observations stimulated attempts in many laboratories to identify the TIP and the genetic basis of tumor formation. Early results pointed to the possibility that tumor induction might be caused by a plasmid or virus, and eventually led to the discovery of megaplasmids that were present only in the virulent strains of Agrobacterium (Zaenen et al. 1974). The plasmids were appropriately named the Ti (tumor-inducing) plasmids. Only a small part of the plasmid, T-DNA, was shown to be responsible for tumor formation (Chilton et al. 1977) and was found to be present in the nuclear DNA fraction of tumor cells (Chilton et al. 1980; Willmitzer et al. 1980).
The fact that a part of the Ti plasmid was transferred and integrated into the plant genome during tumor formation (transformation), suggested that the plasmid could be used as a vector to transfer other genes. Thus methods were developed to insert DNA (genes) into the Ti plasmid (van Haute et al. 1983; de Framond et al. 1983; Hoekma et al. 1983). Transformed crown-gall tumor tissues, which grew on hormone-free media, formed only highly aberrant shoots in culture. This was found to be related to the presence of genes regulating the synthesis of auxin and cytokinin. Deletion of these genes (disarming of the plasmid) produced transformed tissues that required media supplemented with auxin and cytokinin for continued growth and regenerated normal shoots and plants. These findings led to the use of the Ti plasmid of Agrobacterium as a vector for plant transformation, and kanamycin resistance genes for selection of transformed cells, followed by the regeneration of transformed plants (Bevan et al. 1983; Fraley et al. 1983; Herrera-Estrella et al. 1983; de Block et al. 1984; Horsch et al. 1984, 1985). These results were first presented by three independent research groups on January 18, 1983, at the Miami Winter Symposium, and marked the beginning of the modern era of plant biotechnology.
Methods for the direct delivery of DNA into protoplasts were developed during the early 1980s (Paszkowski et al. 1984; Shillito 1999), especially for the economically important cereal crops as they were considered at the time to be outside the host range of Agrobacterium, and therefore not amenable to Agrobacterium-mediated transformation (see Vasil 1999, 2005). The procedure involved delivering osmotic or electric shock to protoplasts suspended in solutions containing DNA, followed by plating on selection media for the preferential growth of transformed colonies, and eventually plants. Transformation of protoplasts isolated from embryolgenic cell suspension cultures led to the production of the first transgenic cereals (Rhodes et al. 1988; see also Vasil and Vasil 1992). The use of protoplasts for genetic transformation became less attractive once it was shown that monocots, including the cereals, could be transformed by co-cultivation of embryonic tissues or embryogenic cultures and super-virulent strains of Agrobacterium in the presence of acetosyringone, a potent inducer of virulence genes (Hiei et al. 1994; Komari and Kubo 1999).
Technical difficulties with the isolation and long-term maintenance of embryogenic suspension cultures, and limitations of Agrobacterium-mediated transformation, encouraged the search for universal methods of transformation. A wide variety of methods, including those that do not require the use of tissue cultures, were proposed as a means to transform plants (Ledoux and Huart 1969; Doy et al. 1973; Hess et al. 1976, 1990; Pandey 1975; Grant et al. 1980; de Wet et al. 1985; Graves and Goldman 1986; Ohto 1986; de la Pena et al. 1987; Luo and Wu 1988; Zilberstein et al. 1994; Zhao et al. 2006). None of these have been independently confirmed and have, therefore, remained outside the mainstream of plant transformation research.
The novel biolistics procedure, a universal method of plant transformation, was developed by Sanford et al. (1987; Sanford 2000). It involves the high velocity bombardment of DNA-coated gold or tungsten microprojectiles into intact cells or tissues. The Agrobacterium and biolistics procedures, in combination with embryogenic cultures, are the two most widely used methods for plant transformation. The preference of one over the other depends more on the biases of the individual researcher than any inherent differences between the procedures (Altpeter et al. 2005).
Advances in the genetic transformation of plants were dependent on parallel advances in plant molecular biology, the sequencing of plant genomes, and the identification and characterization of many agronomically important genes, leading to the production of a variety of transgenic crops that are resistant to biotic and abiotic stresses, and have improved nutritional qualities. Transgenic crops were first planted commercially in 1996, and have since been grown (mostly canola, cotton, maize, and soybean) on more than 1.4 billion acres in 22 countries (see James 2006). They have contributed more than US$ 23 billion to the economies of developing as well as developed countries (nearly 90% of the transgenic crops are planted by resource-poor farmers), have reduced the use of agro-chemicals, have increased productivity, have improved human health, and have had a positive influence on conservation of the environment and biodiversity. These are truly remarkable achievements of a comparatively new technology, which is expected to profoundly influence international agriculture and food security in the 21st century.