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.