Van Hooff, Peter Jan
(2007)
Genetic manipulation of glutathione levels in lettuce: crop performance and resistance to tipburn.
PhD thesis, University of Nottingham.
Abstract
The four lettuce (Lactuca saliva) cvs. Evola, King Louie, Pic and Robusto were transformed with the binary expression construct pAFQ70.1, using Agrobacterium tumefaciens strain AGL 1. The construct carried the glutathione metabolic genes gshi, gshlI, phgpx and gorI. Both the gshI and gshII genes were fused to sequences encoding the pea glutathione reductase transit peptide (grIp), and were intended to influence glutathione synthesis and metabolism in the chloroplasts.
Reverse transcriptase PCR analysis of cvs. King Louie, Pic and Robusto T0 transformants revealed that expression of the transgenes followed a varied pattern. These variations were most likely due to post-transcriptional gene silencing created by the presence of strong promoters, homology with endogenous plant genes and presence of multiple genes in a single vector. The presence of the transgenes, gshI, gshII, phgpx, and gorI, in cv. King Louie T3 homozygous lines was confirmed by genomic DNA dot blots and Southern blots.
It was hypothesized that transformants would have an increased glutathione pool in the chloroplasts and thus would be able to withstand the damaging effects of active oxygen species generated by environmental stresses. Leaves of transgenic homozygous T3 cv. King Louie lines were shown to have a 2-fold greater glutathione concentration than their respective azygous counterparts, although total antioxidant activity was similar in all lines. However, this did not result in enhanced stress tolerance, with the homozygous lines exhibiting no physiological or morphological advantage compared to the azygous and wild-type lines when grown under saline stress (150 mM NaCl).
Glasshouse trials during the summer of 2005 determined the susceptibility of cv. King Louie T3 homozygous, azygous and wild-type lines to the foliar, stress related disorder tipburn. Wild-type plants grown both under calcium deficient conditions at the University of Nottingham and in a trial at Elsoms Seeds Ltd., Spalding, UK, had a reduced incidence of tipburn compared to transgenic plants of the homozygous and azygous lines. Macroscopic of tipburnt leaves revealed the condition formed sporadically, with small dark sunken necrotic spots spreading along the leaf margin restricting leaf expansion. Microscopic transverse sections of tipburnt leaves showed total collapse, disintegration and necrosis of the leaf structure.
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