Genetic Engineering and Ecology: the Role of Alkaloids in Defense Against Insect Herbivory in Cathatanthus roseus (Madagascar Periwinkle)

EPA Grant Number: GF9501261
Title: Genetic Engineering and Ecology: the Role of Alkaloids in Defense Against Insect Herbivory in Cathatanthus roseus (Madagascar Periwinkle)
Investigators: Shonle, Irene
Institution: University of Chicago
EPA Project Officer: Broadway, Virginia
Project Period: June 1, 1995 through January 1, 2000
Project Amount: $34,000
RFA: STAR Graduate Fellowships (1995) Recipients Lists
Research Category: Academic Fellowships , Ecological Indicators/Assessment/Restoration , Fellowship - Ecology


The objective of this experiment is to further the understanding of the insect protection afforded by alkaloids in the Madagascar Periwinkle, Catharanthus roseus. The experiment will examine the role of the entire class of monoterpenoid indole alkaloids, as well as both early- pathway and late-pathway alkaloids, by examining the consequences of their removal with antisense RNAs for key enzymes. This will provide evidence for their role in a more precise way than achievable by ordinary methods, and will entail one of the first applications of transgenic studies in evolutionary ecology. An understanding of the biological effects of alkaloids is a prerequisite to the genetic engineering of potent allelochemicals into plants. Genes controlling a number of enzymes have been cloned in efforts to understand the regulatory mechanisms of alkaloid synthesis, including the gene for the key enzyme strictosidine synthase (SS) which catalyzes the committed step for alkaloid synthesis in Catharanthus. This enzyme has been over expressed in transgenic cell cultures, as well as transgenic tobacco, using the soil-bacteria Agrobacterium tumefaciens as a transformation vector. Since both SS and tryptophan decarboxylase (TDC) have been shown to be coded by single-copy genes in Catharanthus, antisense RNA techniques are likely to be effective. A further advantage of Catharanthus is that there are few other systems in which multiple enzymes for one pathway have been cloned. Two different cultivars of Catharanthus roseus will be sown in the greenhouse under the established temperature and 16 hour per day regime for use in genetic transformation. 100 plants will be transformed with Agrobacterium containing the anti-sense strictosidine gene, and 100 clones of the same plants will be used as controls. The project will rely on standard cocultivation times and procedures in order to perform plant transformation, and will use the callus-inducing and shoot producing media to regenerate plants. In addition, vacuum infiltration transformation will be attempted. Plants will be placed upside down in Agrobacterium-laden infiltration media, and vacuum pressure applied for 20 minutes. Seeds from these plants will be plated onto germination media. In the lab bioassays, a known number of herbivores will be introduced into a cage with a choice of control plants, plants with no alkaloids, and plants with early-pathway alkaloids only. The amount of leaf area removed will be measured for each plant. In addition, parameters such as weight and development of the insects will be monitored. This will allow for the comparison of the efficacy of early and late alkaloids in the protection of the plant from insects. Genetic engineering enables a detailed understanding of the role of alkaloids in the protection of plants from insect herbivory.

Supplemental Keywords:

RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Ecology, Environmental Chemistry, Genetics, Ecosystem/Assessment/Indicators, Ecosystem Protection, exploratory research environmental biology, Ecological Effects - Environmental Exposure & Risk, Ecological Risk Assessment, Ecology and Ecosystems, Molecular Biology/Genetics, Ecological Indicators, Cathatanthus roseus (Madagascar Perwinkle), insect herbivory, Botany, genetic engineering, ecosystem, biological effects