Particle Bombardment

In 1987, a novel method for plant transformation was introduced by John Sanford and colleagues (37). This was based on a device that used gunpowder to accelerate small tungsten particles to a velocity of approximately 400

m s-1. The particles were coated with DNA and could penetrate plant cells without killing them. Initial experiments showed that this device was capable of delivering tobacco mosaic virus (TMV) RNA into onion epidermis cells (resulting in virus coat protein synthesis) and similarly DNA comprising the CaMV 35S promoter and the cat reporter gene could be delivered, resulting in high levels of transient CAT activity. Later experiments showed that particle bombardment was an efficient method for stable integrative transformation (38). A different device was developed by Agra-cetus Inc., which used a high-voltage electrical discharge to generate the accelerating force (Accellâ„¢) (39). The rather erratic gunpowder-based device was refined to a system based on high-pressure blasts of helium (40), and this is the only commercially available particle gun, which is marketed by Bio-Rad.

A. Advantages and Disadvantages of Particle Bombardment

Particle bombardment is simple both conceptually and in practice. Typically, plasmid DNA is prepared by standard methods and precipitated onto tungsten or gold particles using CaCl2. Spermidine and PEG are included to protect the DNA during precipitation, and the particles are washed and suspended in ethanol before drying onto Mylar aluminized foil. This is fired against a retaining screen that allows the microprojectiles through, to strike the target tissue. Particle bombardment is widely used because it circumvents two major limitations of the Agrobacterium system. First, it is possible to achieve the transformation of any species and cell type by this method because DNA delivery is controlled entirely by physical rather than biological parameters. The range of plant species transformable by particle bombardment is therefore restricted only by the competence of cells for regeneration, and the technique is genotype independent and thus useful for the transformation of elite cultivars as well as model varieties. However, careful optimization is required to tailor the method for different species and different cell types and to achieve the highest efficiency transformation with the least cell damage. Important parameters include acceleration method, particle velocity (controlled by the discharge voltage and/or gas pressure), particle size, and the use of different materials (tungsten, gold) (reviewed in Ref. 41). It has also been shown that, for some species and/or tissues, osmotic pretreat-ment prior to bombardment increases the transformation efficiency (42). This can be achieved by partial drying or the addition of osmoticum (mannitol and/or sorbitol) to the culture medium. Optimization experiments are usually carried out by bombarding explants with a screenable marker gene such as gusA and assaying for transient expression (41). In general, stable transfor mation by any direct DNA transfer method occurs at a much lower frequency than transient transformation. A number of different reporter genes are used for transient expression analysis in plants as shown in Table 4.

Second, particle bombardment allows the stable and heritable introduction of many different genes at once using different plasmids, as these tend to concatemerize to form one DNA cluster that integrates at a single locus. Conversely, multiple transformation using the Agrobacterium system requires the cointegration of all the genes in the same T-DNA. Chen et al. (43) reported the cotransformation of rice with 14 separate plasmids containing various marker genes and showed data confirming the cointegration of at least 13 of the plasmids in one plant. Cotransformation has also been used to introduce up to four agronomically important genes into rice plants, producing plants showing resistance to a spectrum of insect pests and plants with pyramiding resistance against individual pests (44,45). Cointegration prevents different transgenes segregating at meiosis. This can be very important in breeding programs where plants carry two or more transgenes required to generate a single protein, e.g., in plants expressing recombinant human antibodies.

A potential disadvantage of the particle bombardment method is the cost of purchasing or hiring the bombardment device. However, a number of articles have been published providing instructions for the construction of alternative economical devices, such as the particle inflow gun based on flowing helium (46). Another disadvantage of particle bombardment is the tendency for DNA sequences introduced by this method to undergo complex rearrangements prior to or during integration. Transgene rearrangement is a pitfall of all direct DNA transfer methods but is perhaps more acute in particle bombardment because the forces involved may cause more DNA fragmentation than other methods and because bombarded plant cells may be induced to produce DNA degradation and repair enzymes in response to their injury (47). This may limit the usefulness of particle bombardment for the introduction of large DNA molecules. However, although an upper size limit has not been established, it has been possible to introduce YAC complementary DNA (cDNA) clones into plant cells by this method (48).

B. Recent Advances

Recent advances in particle bombardment technology include the clean DNA and agrolistic systems for limiting the amount of plasmid backbone sequence that enters the plant genome, as discussed in more detail below. Successful nuclear integration requires that the metal particle actually enters the nucleus (49), and recent experiments have shown that transgene integration may be facilitated by damage caused to DNA strands as the particle moves through

Table 4 Reporter Genes Used in Plants

Reporter gene (product)

Comments gusA (/3-glucuronidase)

cat (chloramphenicol acetyltransferase)

luc (luciferase)

Anthocyanin regulators

Source: E. coli

Activity: catalyzes the hydrolysis of

/3-glucuronides Assays: nonisotropic; in vitro assays are colorimetric or fluorometric; also histochemical assay format using X-gluc

Advantages: simple, sensitive, quantitative, many assay formats available, inexpensive Disadvantages: assays are destructive; enzyme is stable so unsuitable for studies of down-regulation Source: E. coli7r\9

Activity: catalyzes the transfer of acetyl groups from acetyl coenzyme A to chloramphenicol Assays: in vitro assays only, isotropic Advantages: simple to perform Disadvantages: low sensitivity, expensive, low resolution in vivo, reliance on isotopic assay format Source: The firefly Photinus pyralis Activity: light produced in the presence of luciferase, its substrate luciferin, oxygen, Mg2+, and ATP Assays: nonlsotopic bioluminescent assays in vitro and in vivo Advantages: sensitive, rapid turnover, quantitative Disadvantages: expensive detection equipment, limited reproducibility of some assay formats Source: Zea mays Activity: induces pigmentation Assays: visual screening for pigmented cells in vivo Advantages: simple, inexpensive, nondestructive Disadvantages: low sensitivity, not quantitative, background expression, adverse effects on transgenic plants

Table 4 Continued

Reporter gene (product) Comments

Source: the jellyfish Aequorea victoria Activity: intrinsic fluorescence under blue-UV light Assays: nonisotopic, in vivo assays in live plants

Advantages: intrinsic activity (no substrate requirements), sensitivity, use in live plants

Disadvantages: weak signal in some systems (this is being addressed through the use of modifed GFPs with stronger emission and emission at different wavelengths)

Examples of GFP and GUS activity are shown. Source-. Adapted from Refs. 100 and 101.

the nucleus. Occasionally, exogenous DNA integrates at two different sites separated by megabase pairs of DNA (although these still segregate as a single locus). Confocal analysis of the interphase nucleus following fluorescence in situ hybridization (FISH) to transgene sequences has shown that these sites may occupy the same region of the nucleus at interphase and that such integration patterns may reflect localized damage to DNA caused by the particles (author's unpublished data, in preparation).

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