Biolistic method

Some agronomically important plant species (e. g. soybean and most cereal grains) are recalcitrant to Agrobacterium transformation, and a biolistic method (microprojectile bombardment) is frequently used for these plants [47]. DNA coated on micron-sized gold particles is propelled into plant cells using compressed helium gas... 

Wild t3q)e rbcL sequences were introduced into tobacco Sp25 leaves by the biolistic method, and the transformed plants were regenerated on sucrose-containing medium... 

There are several methods that can be used to introduce foreign genes into plant cells, a process called, in general, transformation. Among the most common plant transformation methods are biolistics and exposure to Agrobacterium tumefaciens. 

Biolistics involves bombarding plant cells with tiny (4- am) microprojectiles made of gold or tungsten. These microprojectiles are coated with DNA and are propelled at high velocity from a particle gun or gene gun into plant tissue or cells. In this method, the projectile penetrates the cell wall and carries the transgene into the cell nucleus. 

Transformation of protoplasts by PEG has been demonstrated to work well for chloroplasts as well as plant nuclei. Since chloroplasts tend to be appressed to the cell membranes in protoplasts, they can be penetrated easily by this method. However, this approach is technically more demanding than biolistic delivery and involves a considerably longer period of time for the selection process (Heifetz, 2000). 

Finally, we tested the particle bombardment method, a biolistic approach that was shown before to work when other approaches have failed. In particular, particle bombardment has allowed the generation of transgenic plants such as crop species, which are not susceptible to Agrobacterium tumefaciens or cannot be regenerated from protoplasts. Furthermore, particle bombardment has facilitated organelle transformation in intact cells and the genetic modification of cultured cells that were not accessible to other transfection techniques. 

When we started to elaborate the particle bombardment technique for S. mansoni, two questions were asked. First, is it generally possible to use the biolistic approach to genetically modify schistosomes Second, if yes, does the method allow the generation of reliable and reproducible results To approach these questions, we first designed test vectors for transformation experiments. These vectors... 

Sanford, J.C., Smith, F.D. and Russel, J.A. (1993) Optimizing the biolistic process for different biological applications. Methods in Enzymology 36, 483-513. 

Oliveira, S.C., Harms, J.S., Rosinha, G.M., Rodarte, R.S., Rech, E.L. and Splitter, G.A. (2000) Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host. J. Immunol. Methods, 245, 109-118. 

This technique, also referred to as biolistics (biology + ballistics), is a direct DNA delivery method developed by Sandford and co-workers [16-18]. This technique employs microprojectiles or high velocity particles coated with DNA to deliver exogenous genetic material into the target cell or tissue, which is subsequently in vitro cultured, and regenerated to produce mature transformed plants. 

There are different techniques to overcome the cell membrane barrier and introduce exogenous impermeable compounds, such as dyes, DNA, proteins, and amino acids into the ceU. Some of the methods include lipofection, fusion of cationic liposome, electroporation, microinjection, optoporation, electroinjection, and biolistics. Electroporation has the advantage of being a noncontact method for transient permeabilization of cells (Olofsson et al., 2003). In contrast to microinjection techniques for single cells and single nuclei (Capecchi, 1980), the electroporation technique can be applied to biological containers of sub-femtoliter volumes, that are less than a few micrometers in diameter. Also, it can be extremely fast and well-timed (Kinosita et al., 1988 Hibino et al., 1991), which is of importance in studying fast-reaction phenomena (Ryttsen et aL, 2000). 

Various techniques for neuronal transfection have been reported including viral transfer, DNA/calcium-phosphate coprecipitation, electroporation, microinjection, biolistics, and liposomes. Although the ideal transfection method has not been developed, we describe a neuronal transfection method with LF2000, a commercially available cationic lipid reagent. This method yields consistent transfection efficiency without any time consumption, experienced culture technique, or cell toxicity. 

Hand-held devices similar in principle to the original Biolistics and AcceU instruments are also in use. The most widely-used device is the Biolistics apparatus marketed by Bio-Rad, Inc., but Accell-based methodology has been particularly useful in developing variety-independent gene transfer methods for the more recalcitrant cereals and legumes. Detailed descriptions of the various acceleration devices, principles of operation, and other details may be found in the primaiy references. 

FIGURE 14.1 The Agrobacterium tumefaciens-medmtod and biolistic transformation methods in plants. 

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