Examples of transgenic crop plants expressing herbicide resistance. a. Soybean (Bialaphos); b. Cotton (Bialaphos); c. phaseolus (Bialaphos); d. soybean field trial for resistance to the herbicides Glyphosate and Bialaphos; and e. rice (Bialaphos).
Published in Probe Volume 2(2): Summer 1992
Paul Christou, Ph.D, Senior Scientist,and Dennis
McCabe, Ph.D., Senior Scientist and Research Fellow
Agracetus Inc.
Middleton, WI
The ability to deliver foreign DNA directly into regenerable cells, tissues, or organs appears to provide the best method, at present, to achieve truly genotype-independent transformation in many agronomic crops, bypassing Agrobacterium host-specificity and tissue culture-related regeneration difficulties. Microprojectile bombardment employs high-velocity metal particles to deliver biologically active DNA into plant cells. This concept has been described in detail by Sanford (1988). Following the original observation by Klein et al. (1987) that tungsten particles could be used to introduce macromolecules into epidermal cells of onion with subsequent transient expression of enzymes encoded by these compounds, Christou and McCabe (Christou et al., 1988) demonstrated that this process could be used to deliver biologically active DNA into living cells and produce stable transformants.
Combining the relative ease of DNA introduction into plant cells with an efficient regeneration protocol, which does not require protoplast or suspension cultures, particle bombardment is the optimum system for transformation. Important advancements and refinements using soybean (McCabe et al., 1988; Christou et al., 1990), corn (Gordon-Kamm et al., 1990; Fromm et al., 1990), and rice (Christou et al., 1991) as model systems for dicots and monocots demonstrated the power and versatility of the technique.
Methods Used
Different methods have been used to accelerate particles into living cells (Sanford et al., 1987). These include pneumatic devices; instruments utilizing a mechanical impulse or macroprojectile; centripetal, magnetic or electrostatic forces; spray or vaccination guns; and apparatus based on acceleration by shock wave, such as electric discharge. Metal particles may be coated with DNA or RNA, or they may be used to carry the genetic material into a cell from a solution of DNA or RNA surrounding the cell.
Several variables have been identified and need to be carefully considered in experiments involving transformation utilizing particle bombardment. Physical parameters include (1) the nature, chemical, and physical properties of the metal particles; (2) the nature, preparation, and binding of DNA onto the particles; and (3) the characteristics of the target tissue.
Environmental variables include such parameters as temperature, photoperiod and humidity of donor plants, explants, and bombarded tissues.
Biological factors include choice and nature of explant, pre-and post-bombardment culture conditions, and interactions between the introduced DNA and cytoplasmic or nuclear components.
ACCELL™ Technology
To address the majority of the variables identified as critical to the transformation process, the ACCELL™ gene delivery method was developed to permit maximum flexibility for tuning, targeting, and cell penetration. By varying the intensity of an electric discharge through a water droplet (thus creating a shock wave accelerating the DNA-coated gold particles), penetration of the target tissue can be controlled very accurately. Using this mechanism, the majority of the particles carrying the DNA can be directed to a specific cell layer. This capability is extremely important as even identical explants from different genotypes of the same species may require different acceleration conditions for optimum particle penetration.
Examples of transgenic crop plants expressing herbicide resistance. a.
Soybean (Bialaphos); b. Cotton (Bialaphos); c. phaseolus (Bialaphos); d.
soybean field trial for resistance to the herbicides Glyphosate and
Bialaphos; and e. rice (Bialaphos).
Hundreds of independently derived soybean plants transformed by this method have maintained the foreign genes for many generations. Elite soybean varieties expressing resistance to the herbicides Basta and Roundup , engineered through particle bombardment using electric discharge, are currently undergoing large-scale field evaluation. In a parallel series of experiments, similar approaches resulted in the development of variety-independent transformation protocols for cotton (McCabe and Martinell, 1991).
Transgenic Rice Plants
Until recently, recovery of transgenic rice plants was possible only by using direct DNA transfer methods such as electroporation (e.g., Toriyama et al., 1988) or PEG-mediated transformation (e.g., Datta et al., 1990) of protoplasts. Although genetic engineering of rice had been reported, very few cultivars could be transformed using protoplast-dependent methods. ACCELL™ technology was successfully used to transform immature rice embryos (Christou et al., 1991). Transgenic plants expressing marker genes in addition to antibiotic-, herbicide-, and insectresistance genes were obtained at frequencies >2 percent. When progeny from transgenic rice plants carrying the bar gene were sprayed with the herbicide Basta, they were shown to express total resistance to the herbicide at levels of 2000 ppm, whereas non-transgenic plants were effectively killed at levels of 250- 500 ppm.
Particle bombardment is certainly not a panacea; major technical and scientific barriers need to be overcome to bring the technology to its full potential. It is clear, however, that utilization of this technology opens the way for effective gene transfer into tissues and species that are otherwise inaccessible to genetic modifications using recombinant DNA techniques. Genetic engineering of such recalcitrant crops as those mentioned above is now possible and in some cases routine. Soybean and cotton plants that are highly resistant to commercial herbicides and insect pests will be some of the first agricultural commercial products of recombinant DNA technology. These plants are expected to be on the market well before the end of the decade (Cutler, 1991).
Additional Applications
In addition to the transformation of recalcitrant agronomic crops, woody species have been engineered using this technology. These include poplar (McCown et al., 1991), cranberry (Serres et al., 1991), and spruce (Ellis et al., 1991). A number of additional applications of particle bombardment have been recognized and are currently in use. These include transient expression studies, mechanical viral infections, gene deletion and promoter analyses, organelle and microorganism transformations, studies of basic plant development, introduction of multiple genes into plants, RNA delivery, studies of biosynthetic pathways in plants, and mammalian cell and organ transformations.
References
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