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**Novel tools** To increase the flux through a pathway for secondary metabolite production it may be desirable to block a competitive pathway by the use of antisense or sense (co-suppression) constructs. However, this usually doesnot lead to complete inactivation. A gene disruption would however l...

**Novel tools** To increase the flux through a pathway for secondary metabolite production it may be desirable to block a competitive pathway by the use of antisense or sense (co-suppression) constructs. However, this usually doesnot lead to complete inactivation. A gene disruption would however lead to a complete loss of gene activity. **Transposons** form a useful tool to accomplish this. If these are not present naturally in the host plant of interest, they can be introduced from a heterologous source. The transposon systems from maize have been introduced into several other plant species to induce transposon-tagged mutations. Mutants can be identified by phenotype. Reversely, one can screen in libraries of transposon-tagged mutants, which are available for arabidopsis and petunia, for insertions in the gene of interest by PCR, and thus identify individuals in which there is a mutation in this gene. A similar strategy has been used to identify mutants with a T-DNA insertion in the gene of interest in a large collection of T-DNA insertion mutants of Arabidopsis thaliana. However, it will be feasible only for a few plant species to assemble such a large collection of insertion mutants. **Gene targeting** might become an alternative especially for plants for which such collections are not readily available. Gene targeting is the directed integration of transgenes at a preferred position in the genome by homologous recombination. This has been used to disrupt or modify genes not only in prokaryotes and unicellular eukaryotes such as yeast, but also in mammals such as the mouse. Transgenic plants often contain T-DNA constructs in which not only the transgene of interest is present, but also certain selection markers for instance. This may be an undesired situation, since it prevents the use of the same selection marker in renewed transformation of the already transgenic line or to perform crosses with other transgenic lines carrying the same selection gene. Also for public acceptance in the market it may be beter if these selection genes are absent from transgenic crops. Recently, a method was developed by which such markers can be deleted from the genome. The method uses **site specific recombination systems** from bacteria (Cre, lox system) or yeasts (FLP, frt system). The genes to be deleted from the plant genome are surrounded by the recognition sequences (30-50 bp sequences) of such a site specific recombiaase. If these sequences are in a direct repeat orientation the sequences in between can be deleted by the recombinase expressed from a plant promoter. The recombinase gene can be introduced or expressed transiently in the transgenic plant from which the selection genes have to be deleted, or can be introduced by crossing with a plant in which this gene is stably present already. Another possibility to obtain marker-free plants was recently suggested. Here the ipt gene, which is naturally present in the T-DNA and which determines cytokinin production, is present within the Ac transposon located in the binary vector T-DNA78. Presence of ipt induces shoot formation. Selection now can be on shoots which are formed in the absence of cytokinin in the culture medium. These abnormal (due to the presence of the ipt gene) shoot lines regenerate normal shoots spontaneously, which lack the ipt gene due to the loss of the Ac element. Although much has been achieved during the last fifteen years in the refinement of the Agrobacterium system as a tool for plant transformation it will be clear that further modifications and assets are needed to comply with specific demands. It is in my mind without doubt that future developments will turn this vector system even more sophisticated and versatile than it is already today. PARTICLE GUN METHODOLOGY AS A TOOL IN METABOLIC ENGINEERING Direct DNA transfer procedures, particularly particle bombardment, offer unique advantages over conventional means such as Agrobacterium for many reasons. The ability to deliver foreign DNA directly into regenerable cells, tissues, or organs appears to provide the best method, at present, for achieving truly genotype-independent DISTINCTIVE METABOLIC AND GENETIC CHARACTERISTICS OF PLANTS **A. Compartmentation** B. Tissue Organization C. Cosuppression #### D. Metabolic Grid #### E. Transformation Procedures *\ Agrobacterium-mediated transformation.* Stable transformation is dependent on several factors,the most important being the plant species to be transformed and the transformation protocol used. An attenuated soil-borne pathogen, *Agrobacterium tumefaciens,* is the most commonly used vector to transform numerous dicotyledonous (broad leaf) plants,including familiar fruits and vegetables such as tomatoes,mustards,and beans (Zupan *et al.,* 2000). This method takes advantage of the ''natural'' plant genetic transformation system that evolved in *Agrobacterium*. Wild-type *Agrobacterium* transfers a segment of DNA (called the T-DNA) from its large tumor-inducing (Ti) plasmid through the plant membranes and incorporates it into the genomic DNA of plant cells adjacent to a wound site. The T-DNA is bounded by 25-bp direct repeats called border sequences,and it contains genes that encode enzymes that direct the commandeered plant cells to produce peculiar amino acids called opines that cannot be catabolized by the plants themselves,but that can be used as primary sources of carbon and nitrogen by the cohabiting bacteria. The T-DNA also includes genes that direct the plant cells to produce plant hormones such as cytokinins, which promote cell division and tumor formation,providing a steadily increasing supply of nutrients for the bacteria. To enable technologically useful plant transformation,the *Agrobacterium* oncogenic hormone biosynthetic genes in the T-DNA have been removed from attenuated bacteria and replaced with multicloning sites where genes of interest as well as dominant selectable markers can be integrated. *Agrobacterium* harboring such recombinant Ti plasmids can then be introduced onto wounded tissues (e.g.,leaf explants in culture) or even directly onto mature plant organs (see below) and the bacterium will transfer the modified T-DNA to some of the cells of the host plant. The wild-type Ti plasmid is very large (200 kb) and is difficult to manipulate. Its utility has been improved by the development of binary vectors (Bevan,1984). In such a system,the Ti plasmid of *Agrobacterium* has been disarmed, i.e.,the T-DNA has been removed but the *vir* regions have been left intact. A separate plasmid that can replicate in both *Escherichia coli* and *Agrobacterium* (hence the term ''binary vector'') is then used. The binary vector carries an origin of replication that is compatible with the Ti plasmid in *Agrobacterium*. This plasmid also carries an artificial T-DNA region into which different transgenes may be introduced. Thus,when the binary vector is introduced into *Agrobacterium* the *vir* genes from the disarmed Ti plasmid will act *in trans* to transfer the recombinant T-DNA from the binary vector to the plant cell. As the binary vectors are smaller and much easier to manipulate than intact Ti plasmids,this tool makes *Agrobacterium*-mediated transformation much more straightforward In general,with *Agrobacterium*-mediated transformation it is necessary to select and propagate transformed plant cells containing the integrated *Agrobacterium* T-DNA from those few initially transformed cells (Zupan *et al.,* 2000). If the experiment requires cell cultures then this process is relatively straightforward with many dicot plants. However, if complete regenerated plants are required then the process is complicated by the need for tissue culture-mediated plant regeneration. Despite considerable effort,plant regeneration remains difficult,problematic,and time consuming. In some cases,unwanted somaclonal variation has been introduced through the tissue culture regeneration system. Until about 5--8 years ago it was thought that *Agrobacterium* was incapable of infecting monocotyledonous plants, which include lilies,palms,and grains. This has led to the development of other transformation systems such as particle bombardment (Klein *et al.,* 1987) and electroporation (Newell,2000) as a means to transform these plants. However,over the past few years there have been numerous successes with the transformation of monocot plants using *Agrobacterium* (Hansen,2000; Hernalsteens *et al.,* 1984; Schafer *et al.,* 1987). Strains containing supervirulent plasmids have facilitated transformation of some recalcitrant monocot plants. It is believed that the factor that limits transformation success in monocot plants is not transfer and integration of T-DNA into the plant genome but plant regeneration. Often the regeneration rates are poor with monocot plants and this is further reduced under selection during transformation. As the T-DNA is integrated into the genome at random sites,regions flanking the T-DNA exert a strong influence on expression levels, necessitating the recovery of several independent transgenic lines to account for this variability. Recent advances in using more regenerable starting material have led to several successes with monocot plants such as rice,maize,and sorghum. The poor regeneration in monocotyledon species has also led to the development of germ-line transformation strategies discussed below. *Electroporation and particle bombardment.* Electroporation of whole tissues is another transformation method that has been used for the transformation of monocot plants (Newell,2000; Sorokin *et al.,* 2000; Terzaghi and Cashmore, 1997),although there are fewer reports of the use of this procedure in the literature compared to *Agrobacterium*mediated transformation. Other considerations when using this method include problems with plant regeneration and the tendency of the technique to insert multiple copies of the transgene(s) into the plant genome. Particle gun-mediated transformation,often called biolistic transformation,is a commonly used procedure that has its advantages (Klein *et al.,* 1987; Maliga,2001; Ye *et al.,* 1990). This is the most frequently used procedure for transient transformation of tissues and is based on bombarding tissues with microscopic, DNA- coated tungsten or gold particles. As with electroporation,one of the disadvantages of this method is that multiple insertions of the transgene occur and can result in gene silencing and instability of the transgene (Hansen and Chilton,1996) (see below). However,this method is not limited by the species or type of tissues bombarded and is frequently used for transformation of monocot species. Another advantage of this procedure is that its high transformation frequency has facilitated the successful transformation of plastids in tobacco and tomato (Maliga,2001). Transforming the plastid genome instead of the nuclear plant genome may be very advantageous for bioengineering reasons (Daniell *et al.,* 1990). For example, this enables targeting of the gene product to the specific organelle in which it is intended to act. Because the plastid genome is often duplicated severalfold within a single plastid and the plastids are themselves present in high copy numbers within many cell types,plastid transformation can lead to substantial gene amplification. This technique also has the added advantage of not transferring transgenes via pollen as plastids are maternally inherited,which makes dispersal of the transgene easier to control. Germ-line transformation However,all the methods described above require the use of plant tissue culture procedures to be able to regenerate transgenic plants. Germ-line transformation has been touted as a means to overcome this limitation by directly transforming germ-line cells (Tague,2001). Its success has been reported for *Arabidopsis thaliana* and some close relatives,of which flowers were dipped into solutions containing *Agrobacterium* in the presence of surfactants (Tague,2001). Transgenic seeds were produced directly but at low frequency from these dipped flowers without need for tissue culture.