DNA delivery systems

Agrobacterium tumefaciens

This bacterium is a natural transformer of somatic host cells of plants into tumorous crown gall cells. Its ability to transform cells with a piece of DNA was exploited by plant biologists, and now Agrobacterium plays a prominent role in transgenesis of plants. This natural gene transfer system is highly efficient, frequently yielding transformants containing single copies of the transferred DNA which have a relatively uncomplicated integration pattern compared with other transformation procedures.

Its utility was developed from the understanding of the molecular basis of the crown gall disease, namely, the transfer of DNA from the bacterium to the plant nuclear genome during the tumor-formation process. Only a small discrete portion of the ca. 200 kbp tumor-inducing plasmid (Ti) existing in the bacterium is transferred to the plant genome. The transferred DNA, now familiarly referred to as T-DNA, is surrounded by two 25-bp imperfect direct repeats and contains oncogenes encoding enzymes for the synthesis of the plant growth regulators auxin and cytokinin and for the synthesis of novel amino acid derivates called opines. The DNA transfer is mediated by a set of bacterial proteins encoded by genes (vir genes) existing in the Ti-plasmid, which become induced by phenolic compounds released upon wounding of the plant tissue. The key aspect in regard to gene transfer is that none of the T-DNA genes are involved in the transfer process and therefore, any or all of these genes can be removed, mutated, or replaced by other genes, and the T-DNA region can still be transferred to the plant genome.

Direct gene transfer

For some time there was good reason to believe that Agrobacterium tumefaciens was the vector system with the capacity for gene transfer to any plant species and variety. As this was not the case, numerous alternative approaches of ‘direct gene transfer’ have been tested. Most methods of direct gene transfer, such as the introduction of DNA via electroporation, [17–19] PEG-mediated DNA uptake, [20–1] protoplast fusion with liposomes containing DNA, [22] biolistics23 or microinjection, [24] require the regeneration of plants from protoplasts. The recalcitrance of many plant species for efficient regeneration from protoplasts, elaborate protocols and prolonged tissue culture phases, are a disadvantage. Other methods for direct gene transfer in which DNA is introduced directly into tissue or whole plants [25–9] do not require protoplasts.

Biolistics, or acceleration of heavy microparticles coated with DNA, has been developed into a technique that carries genes into virtually every type of cell and tissue. Without too much manual effort, this approach has advantages such as easy handling, regeneration of multiple transformants in one shot and utilization of a broad spectrum of target cells, i.e., pollen, cultured cells, meristematic cells, etc. Using this technique, a number of transgenic crops have been produced. Remarkably some of them correspond to recalcitrant species not readily amenable to infection by Agrobacterium such as oat, [30] sugarcane, [31] maize, [32–3] wheat, [34–5] barley, [36] cotton, [37] banana, [38] and soybean. [39] It is not unreasonable to expect that additional major crops will be engineered using this technology. However, although biolistics has impacted significantly on agricultural biotechnology, it is certainly not a panacea. This technique is inefficient in yielding stable integrative events and most of the transformation events are transient. This makes recovery of large numbers of independently derived transformation events labor intensive and expensive.

Electroporation is one of several standard techniques for routine and efficient transformation of plants from protoplasts. [17, 40–1] This technique refers to the process of applying a high-intensity electric field to reversibly permeabilize bilipid membranes and it may be applicable to all cell types. Discharge of a capacitor across cell populations leads to transient openings in the plasmalemma which facilitates entry of DNA molecules into cells if the DNA is in direct contact with the membrane. Transgenic plants recovered using this technique contain from one to few copies of the transfected DNA, which is generally inherited in a Mendelian fashion.