Helena V. Mathews

Activation Tagging in Tomato Identifies a Transcriptional Regulator of Anthocyanin Biosynthesis, Modification, and Transport

We have developed a high-throughput T-DNA insertional mutagenesis program in tomato using activation tagging to identify genes that regulate metabolic pathways. One of the activation-tagged insertion lines (ant1) showed intense purple pigmentation from the very early stage of shoot formation in culture, reflecting activation of the biosynthetic pathway leading to anthocyanin accumulation. The purple coloration resulted from the overexpression of a gene that encodes a MYB transcription factor. Vegetative tissues of ant1 plants displayed intense purple color, and the fruit showed purple spotting on the epidermis and pericarp. The gene-to-trait relationship of ant1 was confirmed by the overexpression of ANT1 in transgenic tomato and in tobacco under the control of a constitutive promoter. Suppression subtractive hybridization and RNA hybridization analysis of the purple tomato plants indicated that the overexpression of ANT1 caused the upregulation of genes that encode proteins in both the early and later steps of anthocyanidin biosynthesis as well as genes involved in the glycosylation and transport of anthocyanins into the vacuole.

Molecular and cellular evidence of chimaeric tissues in primary transgenics and elimination of chimaerism through improved selection protocols

Transgenic plants of strawberry cultivar Totem were developed by Agrobacterium-mediated transformation using a plasmid vector containing gus and nptII genes. Parallel experiments were carried out with and without repeated subculturing (iterative cultures) for generation of transgenic shoots on selection medium. The selection levels in the non-iterative pathway were kept constant, while in the iterative protocol, stepwise increase of selection pressure was applied at different stages of tissue growth. Rooted transgenic plants obtained via both protocols were outplanted in soil. Random leaf samples of greenhouse-grown transgenics were analysed for the presence of gus gene sequences by Southern hybridization as well as gus expression on leaf and petiole tissues by X-Gluc histological assay. Random leaf samples analysed from individual transgenic events developed under iterative culture were positive for the gus insert as verified by Southern analysis confirming the presence of transgenes and lack of chimaeras. Leaf samples of the transgenic events from the non-iterative protocol were either positive or negative on Southern analysis indicating the chimaeric nature of the transgenic plants. The absence of gus sequences in the transgenic plants grown under the non-iterative protocol reinforced the necessity of iterative cultures along with stepwise increase in selection levels for generating non-chimaeric transgenics in strawberry. The gus expression was highly variable, irrespective of the iterative or non-iterative protocol used for transformation. We conclude that strawberry is highly prone to develop chimaeric transgenics if derived from primary regenerants and that the iterative culture technique effectively converts chimaeras to pure line transgenic plants

Genomic Approach to Altering Phytochemicals in Tomato for Human Nutrition

Phytochemicals are non-nutrient, physiologically active plant components present in relatively small amounts compared to the macronutrients (fats, carbohydrates and proteins). Phytochemicals have recently been the focus of intense research efforts because of their cancer preventive properties. Epidemiological studies have demonstrated that populations consuming phytochemicals through a plant-based diet high in grains, legumes, fruits and vegetables have a markedly reduced incidence of cancer. Only recently have biological scientists begun to identify the mechanism through which phytochemicals reduce cancer risk. Some phytochemicals like the organosulfur compounds in allium vegetables such as garlic and onion, detoxify carcinogens and thus help the body to eliminate them. Others such as carotenoids in yellow, red, and green vegetables, function as antioxidants by scavenging free radicals that can attack and damage cellular membranes and DNA. Lycopene in tomatoes is antoher example of a phytochemical that acts as an antioxidant, and has been shown to be especially effective in preventing prostate cancer.