Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 85 phenomenon in some resistant barley lines - for example, barley lines with the durable wheat Lr34 gene (which is a well-known rust resistance gene) also show leaf tip necrosis as a sort of benign side effect. It’s basically considered a marker of the plant’s immune system being on; it often co-occurs with having strong disease resistance. The vast majority of their transgenic barley lines did not show this trait at all, meaning the issue was extremely limited. Next, they examined whether the transgenic barley became more sensitive or susceptible to anything else - like other diseases, pests, or stresses (sometimes enhancing one trait can make another worse). They didn’t find any evidence of increased susceptibility to other pests or diseases. In field observations, the transgenic plants did not show more of other diseases compared to controls. In fact, because they resisted the target diseases, their overall health was better, and they didn’t develop secondary issues. For example, sometimes a plant heavily infected by one disease can then get opportunistically attacked by another, but since the transgenic barley’s leaves stayed clean of mildew and rust, they actually had fewer leaf spots or secondary infections than control plants. Preliminary observations on drought and cold tolerance also showed no difference between transgenics and controls - they seemed to handle abiotic stresses normally, which indicates no trade-off there either. They then looked at reproductive capacity. The transgenic barley had normal flowering, with normal anthers and pollen. Pollen viability tests and seed set rates were comparable to the control. That means the transgenes didn’t cause any fertility issues; the plants could reproduce just fine. The seeds they produced had normal germination and vigor in the next generation (no reduction in seed germination or seedling health was observed in the progeny). This is critical for stable inheritance and also for practical farming (you don’t want a high-yielding disease-resistant plant that produces weak or non-viable seeds). Fortunately, that was not the case - the seeds from transgenic barley were just as good as those from regular barley. Finally, they considered the possibility of a metabolic burden. Producing extra proteins (like chitinase and AMP) might use up some of the plant’s resources - amino acids, energy, etc. If this burden was significant, it might show up as reduced growth or yield, or changes in composition (like lower grain nitrogen because a lot was tied up in making the new proteins). They combined the yield data with analyses of plant nitrogen content and found no evidence that the transgenic plants were “suffering” from making these proteins. Grain nitrogen (protein content) was within normal range, and the total biomass of the plants wasn’t reduced, which suggests that the plants compensated for the production of the antifungal proteins without a problem. It helps that these antifungal proteins are probably not the most massive drain - chitinase and the AMP are significant, but they ended up being maybe around 1% or so of total protein, which plants can often handle. Also, the localization of these proteins mostly in the cell wall and intercellular spaces (for chitinase and secreted AMP) likely means they are not interfering with internal cellular metabolism. They do their work outside the cells, so inside the cells things proceed normally. This is supported by the observation that the transgenics didn’t have altered metabolism signs like changes in growth rate or abnormal accumulations of other compounds. To further confirm there were no toxic effects of the antifungal proteins on the plant’s own seeds or germination process, they ran seed germination tests in the lab. Transgenic seeds germinated just as well as control seeds. If the antifungal proteins were somehow toxic, you might see poor germination or seedling abnormalities, but they saw none of that. This indicates the antifungal proteins did not interfere with seed nutrient mobilization (the seed has to break down its starch and protein to feed the embryo when germinating; since chitinase mainly targets fungal cell walls and the AMP targets pathogens, they don’t have a target in the seed’s own biology - and the results show they didn’t accidentally mess something up either). Bregitzer et al. (2002) and Boni et al. (2017) noted that in other cases where growth problems were observed, these were generally specific. In our case, there was no significant loss of fitness, which is consistent with the findings of Boni et al. (2017) where disease resistance genes did not impair barley growth. Bregitzer et al. (2002) may have discussed avoiding somaclonal variation or similar situations in tissue culture, which they also carefully monitored. In summary, the transgenic barley did not exhibit any significant loss of fitness or abnormal phenotypes. They were as robust, productive, and stress-tolerant as conventional barley, with the added advantage
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