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Barley with the transgene for a heat-stable enzyme showed highly variable levels of the enzyme, an anomalous distribution of expression in the grain, and dramatically reduced weight of individual grains.

Manipulated Organism: Barley (Hordeum vulgare L.), cultivar Golden Promise.

Inserted Transgenes: Gene for producing the heat stable enzyme beta-glucanase, derived from bacteria. Two other genes were also part of the construct: a selectable marker gene (bar) and a reporter gene (uidaA). So that the genes would be expressed in all parts of the plant, they were fused with a constitutive promoter (either maize pUbi-1 or synthetic pEmu).

Goal: Produce barley plants with enhanced ability to break down cell walls during high-temperature malting. The introduced transgene would induce barley to produce the heat-stable enzyme, beta-glucanase, which breaks down cell wall components and does not degrade at high temperatures (over 55° Celsius).

Intended Effect: Three plants contained all three genes (in multiple copies) and from these eighteen homozygous lines of transgenic plants were propagated. These were grown under field conditions and they stably expressed the target enzyme, beta-glucanase, over three years.

Unintended Effects:
  • Although all the transgenic barley plants showed similar DNA fragment patterns (Southern blot analysis), they "produced significantly different amounts of the recombinant enzyme," some having high levels, some low levels, and others intermediate levels of expression.
  • The plants with high levels of beta-glucanase showed no expression of the reporter gene (uidaA), although the gene was present, while in plants with low levels of beta-glucanase, reporter gene expression was "readily detected" (p. 10).
  • The weight of the grains of the transgenic barley plants in 1997 and 1998 was on average 31% and 41% less than unmanipulated barley plants, although overall yield was comparable.
  • The active heat-stable enzyme was not present in the endosperm (nutrient tissue) of the grain, where it "should" have been expressed, but rather in the embryo of the grain, where it was not supposed to be expressed. (Evidently the attached promoter did not function as intended, perhaps due to "unsuitable insertion into chromatin" p. 10.)
  • The performance of high-enzyme-expressing transgenic lines and two normal cultivars was compared under differing field conditions. There was a "dramatic impact of different conditions on the field performance of wild-type cultivars and the transgenic barley lines" (p. 4). Under irrigated conditions in Idaho all types performed best; grain weight and yield was, however, lower in the transgenic lines.

Additional Comments: The authors state that "it is remarkable that plants with so similar patterns in Southern blot analysis are not only different in the expression levels of the analyzed intact beta-glucanase gene but also for GUS [reporter gene (uidaA)] expression" (p. 10).

Source: Horvath, H., L. G. Jensen, O. T. Wong, E. Kohl et al. (2001). "Stability of Transgene Expression, Field Performance and Recombination Breeding of Transformed Barley Lines," Theor Appl Genet vol. 102, pp. 1-11.

Author Affiliations: Washington State University; Plant Biology and Biochemistry Department, Risø National Laboratory, Denmark.

Funding: USDA; Applied Phytologics, Sacramento, California; Washington Technology Center (government/university/industry collaborative).

Product Status: Not on the market as of 2008.

Copyright 2008 The Nature Institute.

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