Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 81 control barley. In bad FHB outbreak years, the control barley had over 20% of its spikes infected, resulting in lots of shriveled, light “scabby” grains, often with a pinkish-white mold on them, and these grains contained the mycotoxin DON. The transgenic barley, by contrast, fared much better: its grains remained plump and healthy in those same conditions, and tests showed no significant accumulation of DON toxin. In other words, the antifungal proteins seemed to protect the barley from Fusarium infection in the field, keeping the grains sound and safe. In additional plot trials, they also noted that some of the high-expressing transgenic lines showed complete resistance to powdery mildew and leaf rust in the field (corroborating the greenhouse results). The only minor issue observed was a little bit of leaf tip necrosis in some cases. This appears as tiny dead patches at the very tips of leaves. Interestingly, this phenomenon is known in some plants that have very strong disease resistance (it’s seen, for example, in wheat lines carrying the Lr34 durable resistance gene and is considered a kind of hypersensitive response or a mild side-effect of the immune system being active). In the transgenic barley, a slight leaf tip necrosis was seen in some lines but it was not a serious problem and didn’t spread beyond the tips. It’s believed to be a sign of the plant’s heightened immune state rather than a disease. Crucially, no other negative symptoms were observed in the field. The transgenic plants with Chi and AMP genes grew normally, yielded normally, and did not show any issues like lodging (falling over) or any unexpected susceptibility to other problems. This indicates that the level of antifungal protein expression was well-tolerated and didn’t trade off general plant health. Overall, the greenhouse and field inoculation experiments demonstrated that the transgenic barley’s improved disease resistance was real and significant. Under pathogen attack, transgenic plants had far less disease than their normal counterparts - in some cases, virtually none. This shows that the antifungal proteins were functioning effectively inside the plants to ward off infections. 5.3 Disease resistance scoring and statistical evaluation To quantify the disease resistance more formally, the researchers used standardized scoring systems and did statistical analyses on the data from their inoculation experiments. In the greenhouse, they scored powdery mildew severity on a 0-5 scale (0 = no symptoms, 5 = very severe infection covering most of the leaf) and leaf rust on a 0-4 scale (based on rust pustule coverage) (Bollina et al., 2010; Poznański et al., 2023). The average powdery mildew score for the control plants was about 4.0, indicating they were heavily infected (susceptible). Meanwhile, the transgenic lines scored much lower: the chitinase-expressing lines averaged around 1.5, the AMP lines around 2.0, and the dual-gene lines around 1.0. All these differences were statistically significant (P < 0.01) - meaning the improvements in the transgenics were not due to chance. Similarly for leaf rust, the control plants scored about 3.5 on average, whereas the transgenic Chi lines were around 1.0 and AMP lines around 1.5. This again showed that the transgenic plants achieved high resistance or near-immunity to both powdery mildew and leaf rust. For the field Fusarium head blight observation, they calculated the percentage of infected ears and a disease index (which incorporates both the incidence and severity of disease on those ears). The control barley had roughly 25% of ears showing disease, with a disease index around 20. The transgenic Chi line, however, had less than 5% of ears infected and a disease index below 5. The AMP line had a disease index below 10. In both cases, the transgenic lines were significantly better than the control (P < 0.01). This quantifies what we described earlier qualitatively - there was a major reduction in disease in the transgenics. They used analysis of variance (ANOVA) to verify that the differences observed among the different genotypes (control vs various transgenics) were statistically highly significant. Essentially, there’s a very low probability that these differences are just due to random variation; it’s clear they are due to the presence of the antifungal genes. They also looked into the effect of gene stacking (i.e., having both Chi and AMP vs just one). While the dual-gene line performed slightly better than the single-gene lines (as seen in the raw data), when statistically analyzed, the difference between, say, the dual-gene line and the best single-gene line was not significant. One explanation
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