UBC Theses and Dissertations
Transgenic approaches to improved insect resistance in poplar Gill, Rishi Indra Singh
Amines and their derivatives are known to influence insect behavior involved in feeding and reproduction. In order to examine the feasibility of improving the resistance of poplar to insect pests by the introduction of a plant-derived amine-generating transgene, explants from the hybrid poplar clone 'NC5339' (Populus alba x P. grandidentata cv. 'Crandon') and 'INRA 717 1B4' (P. tremula x P. alba) were transformed with a Camptotheca acuminata tryptophan decarboxylase cDNA driven by the CaMV35S promoter. The enzyme tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine, which, in addition to being a bio-active amine itself, is known to act as a precursor of various other indole derivatives. TDC1 transgenic tobacco plants were also generated, to allow a comparison of the TDC1 overexpression phenotype in both a herbaceous and a woody perennial species. Poplar and tobacco plants were also transformed with an 'empty' vector (pBin19/PRT101) to use as a control in insect bioassays. Putative transgenic lines were confirmed by PCR for the TDC1 gene sequence and by the expression analysis of the transgene mRNA and encoded protein. Chemical and radiotracer analyses of the transgenic plants suggested that the primary product of tryptophan decarboxylation was tryptamine, which was not metabolized further. No visible phenotypic changes were associated with ectopic TDC1 expression in either species. In insect bioassays, an increased accumulation of tryptamine in TDC1 transgenic lines was consistently associated with an adverse effect on feeding behavior and physiology of Malacosoma disstria (forest tent caterpillar, FTC) and Manduca sexta (tobacco hornworm, THW). Behavior studies with FTC and THW larvae showed that the acceptability of the leaf tissue to larvae was substantially reduced as the tryptamine levels in the tissue increased. Physiological studies with the FTC and THW larvae showed that consumption of leaf tissue from the transgenic lines is clearly deleterious to larva growth. The growth inhibition induced by feeding on high TDC1 tissue is presumably due to a post-ingestive physiological mechanism. This work demonstrates that ectopic expression of TDC1 can allow sufficient tryptamine to accumulate in poplar and tobacco leaf tissue to adversely affect the growth of insect pests that normally feed on these plants.
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