Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 80 Additionally, the results hinted at a potential synergistic benefit when multiple antifungal proteins were combined in one plant (as seen by the high efficacy of the dual Chi+AMP extract). The researchers did caution, however, that results from these high-concentration protein assays in vitro may not directly mirror what happens in a living plant, where protein concentrations and environmental interactions differ. Therefore, while these in vitro results strongly suggested that the transgenic barley’s proteins could inhibit fungi, the ultimate proof would come from in vivo tests - infecting the actual plants and seeing if they resist disease. 5.2 Pathogen inoculation in greenhouse or field After the encouraging test-tube and petri dish results, the team moved on to inoculation experiments on the actual plants, both in greenhouse conditions and in the field, to evaluate disease resistance in a real-world context. Greenhouse trials: They focused on two major foliar diseases of barley - powdery mildew and leaf rust. Transgenic barley plants (expressing the antifungal proteins) and non-transgenic control plants were grown under the same conditions in pots. For powdery mildew, when the transgenic and control plants reached the three-leaf stage, the researchers sprayed their leaves with a high concentration of powdery mildew spores (about 10^5 spores/mL) to simulate a heavy infection. Similarly, for leaf rust, they inoculated another set of plants at the jointing stage (when stems are elongating) with leaf rust spores. The greenhouse was kept warm and humid to favor disease development, and they assessed disease symptoms about 10 days and 15 days post-inoculation. The difference between transgenic and control plants was dramatic. The control barley plants developed severe powdery mildew: their leaves were covered in the typical white, powdery fungal growth, with more than half of each leaf’s area showing infection. In contrast, the transgenic barley plants had very few powdery mildew spots - just some small, isolated speckles - and no widespread mildew patches. When they quantified disease severity (using a disease index or scoring system), the powdery mildew index for the transgenic plants was much lower. Specifically, barley lines with the chitinase gene had about 30% of the disease level of the controls, and those with the AMP gene had around 40% of the control’s disease level. Both are a huge improvement over the control. The slight difference between chitinase and AMP lines might be due to the different ways those proteins work (one might be slightly more effective against this fungus than the other), but importantly both kinds of transgenic plants were highly resistant. For leaf rust, the results were similar. Control plants’ leaves got covered in rust pustules - those orange-brown spots full of spores - indicating a heavy infection. The transgenic plants, on the other hand, showed almost no rust. At most, you’d find a rare tiny rust spot on some leaves, but many leaves had none. Quantitatively, if the control had a high rust score (e.g., 3.5 on a 0-4 scale, meaning very susceptible), the transgenic Chi line scored around 1.0 and the AMP line around 1.5, which corresponds to a high level of resistance. The most impressive performance was seen in transgenic plants that had both the Chi and AMP genes. These bivalent lines were practically immune in the greenhouse tests. Under heavy pathogen pressure, the dual-gene plants showed virtually no disease lesions at all. It was as if the pathogens couldn’t establish on them. This broad and robust resistance in the dual-gene line mirrors what has been observed in some transgenic wheat experiments where stacking multiple resistance genes led to very high levels of protection. The success of the dual transgenic barley suggests that combining antifungal mechanisms (cell wall degradation by chitinase+membrane disruption by AMP, for instance) can yield an additive or even synergistic effect against pathogens. The researchers pointed out that this kind of broad-spectrum, high-level resistance is comparable to or better than what’s achieved by some of the best conventional resistance genes. It also resonates with what was seen in wheat when foreign genes were introduced (wheat is another crop where adding new resistance genes sometimes produced almost complete immunity to certain diseases). Field observations: They didn’t stop at the greenhouse; they also tested the transgenic barley in the field, particularly against Fusarium head blight (FHB), which occurs under natural conditions in certain endemic areas. In a field trial set in an area prone to Fusarium, they observed what happened to the grains of transgenic vs.
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