Genetic Engineering

Development of methods to transfer genes of interest into tomato has made it possible to use biotechno-logical approaches for the study of gene function. An essential prerequisite for a gene transfer system is an efficient and reliable method to regenerate plants from cells in various plant tissues. A significant amount of effort by many research groups has gone into development of an efficient regeneration system for tomato. There were several reports prior to the first reports on transformation (Behki and Leslley 1976; Kartha et al. 1976), and improvements on regeneration methods continued even after the first successful transformations were reported (Poonam et al. 2004). Regeneration from in vitro tomato cultures has been shown to be dependent upon the genotype (Stommel and Sin-den 1991), explant type (Poonam et al. 2004), culture medium, namely the plant growth regulators (Uddin et al. 1988), and culture conditions (light, temperature) (Reynolds et al. 1982; Lercari et al. 2002; Poonam et al. 2004).

The earliest reports of Agrobacterium tumefa-ciens-mediated transformation of tomato were approximately 20 years ago (Horsch etal. 1985; Mc-Cormick et al. 1986). During the intervening years, there have been numerous reports of tomato transformation methods and projects that resulted in the recovery of transgenic lines that were successfully transferred to the greenhouse and field for further study. Plant material from sterile, in vitro-grown seedlings has been the primary source of explants for transformation with cotyledon segments being the most commonly used seedling part (Fillatti et al. 1987; Hamza and Chupeau 1993; van Roekel et al. 1993; Ellul et al. 2003; Frary and Van Eck 2004). However, hypocotyls segments from seedlings have also been used in some studies (Frary and Earle 1996; Park etal. 2003), although Frary and Earle (1996) reported that the time for recovery of transformants from hypocotyls took longer than recovery from cotyledon segments. There are also reports of using surface sterilized leaves from greenhouse-grown plants as a source of explants for transformations (Horsch et al. 1985;McCormicket al. 1986) and leaves from sterile, in vitro-grown plants (Sigareva et al. 2004). However, there are fewer reports where leaf segments were used for transformations as compared to the use of cotyledon segments.

When all the components necessary for an efficient regeneration and transformation system are in place, the plant material has the potential to regenerate a large number of shoots. To make recovery of transformants more efficient, a selectable marker gene is often included in the gene constructs used for transformations. These selectable marker genes can confer resistance to either antibiotics or herbicides. Therefore, when the appropriate concentration of either an antibiotic or herbicide component is incorporated into culture medium used for transformations, the non-transformed cells do not regenerate shoots, whereas, the cells containing the selectable marker and gene of interest can continue to grow and regenerate shoots. Various selectable markers have been used successfully for tomato transformation. The most commonly used selectable marker for tomato transformation has been the neomycin phosphotransferase gene (nptII), which was isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. This gene confers resistance to a range of aminoglycoside antibiotics including kanamycin, which is the most commonly used selection agent when nptII is used as the selectable marker gene. Another antibiotic resistance gene that has been used successfully for tomato transformation is the gene for hygromycin phosphotransferase (hpt), which is also from E. coli and confers resistance to the antibiotic hygromycin (Van Eck et al. 2006). Van Eck etal. (2006) also reported the use of the herbicide resistance gene, bar, from Streptomyces hygmscopius as a selectable marker for tomato transformation. The selection agent used for transformations with bar as the selectable marker was bialaphos. With interest in development of selection systems that would be more acceptable by the public, efforts are underway by a number of groups to find selection strategies that are not based on antibiotic or herbicide resistance. One such method was reported for tomato and is based on using a carbohydrate, mannose, as a selection agent. (Sigareva et al. 2004). For this selectable marker system, the phosphomannose isomerase gene (pmi) was used and mannose was incorporated into the culture medium to select for transformed cells. Non-transformed cells in the leaf explants could not grow on the mannose-containing medium, therefore only the transformed cells were able to grow and produce shoots.

For initial investigations of tomato transformation, genes were chosen for purposes of proof-of-concept and development of an efficient and reliable transformation method, rather than their influence on agronomically important traits. Following reports of efficient transformation methods for various genotypes, researchers have investigated traits of interest such as insect tolerance (Fischhoff et al. 1987), ripening (Vrebalov et al. 2002; Xiaong et al. 2005), bacterial disease resistance (Li and Steffens 2002), virus tolerance (Xu et al. 2004), and improved growth under environmental stresses (Hsieh et al. 2002; Jia et al.

2002; Mishra etal. 2002). Currently, there are no genetically engineered tomatoes on the market. However, there are several companies that have been involved or are currently developing genetically engineered tomatoes that will have a commercial application. For additional information on efforts for commercialization of genetically engineered tomatoes see http://www.geo-pie.cornell.edu/ crops/tomato.html.

In addition to interest in traits of agronomic importance, tomato is also being investigated for production of plant-made pharmaceuticals, namely orally delivered vaccines (Van Eck et al. 2006). Characteristics that make tomatoes a good choice for production of orally delivered vaccines include high fruit yield, non-toxicity, reasonable biomass and protein content, and easy containment and production in greenhouses, even at commercial scales. Tomato has been investigated for production of vaccines for respiratory syncytial virus (Sandhu et al. 2000), cholera (jiang et al. 2007), hepatitis E (Ma et al. 2003), and diarrheal illnesses caused by E. coli (Walmsley et al. 2003). Although there is some concern about consistency of amounts produced in each individual fruit, studies are in progress to produce freeze-dried material from batch harvests that can be packaged in capsules for delivery of controlled and effective amounts of the vaccine (Van Eck et al. 2006).

With the advent of genome sequencing for many plant species, functional genomics plays an important role in gene discovery, which requires methods for high-throughput generation of mutant and transgenic lines. Such is the case for tomato, where researchers are already investigating approaches for high-throughput transformation systems in anticipation of available tomato genome sequence. One important component of a high-throughput transformation system for functional genomics studies is the selection of a genotype that is amenable to transformation, has a fast generation cycle, and has a small size so that a large number of plants can be grown without the need for expansive growing areas. A tomato genotype that is being investigated for high-throughput transformation is Micro-Tom, a miniature-dwarf-determinate S. lycopersicum cultivar (Meissner et al. 1997). It can be grown at a high density, yields mature fruit within 70-90 days of sowing and is amenable to Agrobacterium tumefaciens-mediated transformation (Dan et al. 2006; Sun et al. 2006).

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