Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 79 instance, harmed the cells or caused any developmental issues. In fact, they specifically observed that there were no deleterious effects like stunted growth or weird morphology in any tissue, which implies the promoters and signal peptides used were appropriate and not causing harm to the plant (Burton et al., 2011). 5 Functional Assays of Antifungal Activity 5.1 In vitro inhibition assays With the transgenic barley plants in hand and confirmation that they were producing the antifungal proteins, the researchers next wanted to test whether those proteins were actually doing their job - namely, inhibiting fungi. They started with a series of in vitro assays, which are controlled lab tests outside the plant. They used several common methods to evaluate antifungal activity, including inhibition zone assays, microtiter plate assays, and disk diffusion assays. Essentially, these methods involve exposing a fungus to the protein extracts from the plants and seeing if the fungus’s growth is inhibited. To prepare for these tests, they extracted soluble proteins from the leaves of transgenic barley and concentrated them to make a “crude antifungal extract”. For comparison, they did the same with leaves from non-transgenic control barley to serve as a negative control extract. This way, any difference in fungal growth could be attributed to the presence of the antifungal proteins in the transgenic extract. One specific experiment they did was to test the effect on powdery mildew spore germination and appressorium formation. Powdery mildew fungi germinate on the leaf surface and form structures called appressoria to infect the plant. The researchers took spore suspensions of the powdery mildew fungus and mixed them with either the transgenic barley protein extract or the control extract. These mixtures were incubated on microscope slides for about 8 hours, then stained and observed under a microscope to see how many spores successfully formed appressoria. The results were striking: spores treated with the transgenic barley extract had a significantly lower appressorium formation rate - roughly half that of the control group (and this difference was statistically significant, P < 0.01). In particular, the extract from plants that had both the chitinase and AMP genes was the most effective, with over 70% inhibition of appressorium formation relative to the control. This suggests that the chitinase and the antimicrobial peptide were both contributing to stopping the fungus at this early stage (chitinase attacking the spore wall, AMP possibly affecting the germ tube or cell membrane). Essentially, the transgenic extract was preventing many of the fungus’s spores from successfully initiating infection. Next, they tested the effect on the growth of a fungal mycelium, specifically using Gibberella fusca (which is one of the Fusarium species). They set up petri dishes with PDA (potato dextrose agar) growth medium and added the barley protein extracts to the agar. Then they placed equal-sized plugs of fungal mycelium in the center of these plates. On control plates (with extract from normal barley), the fungus grew out rapidly - after five days, the colonies expanded to over 9 cm in diameter, pretty much covering the plate. On plates that contained the transgenic barley extracts, the fungus grew much more slowly: on average, those colonies reached only about 60% of the diameter of the control colonies in the same time frame. Notably, the plates containing the chitinase-rich extract had the strongest effect - the fungal growth was sparse and the colony edges were wrinkled and irregular, which is a typical sign of stress (likely because the chitinase enzyme was degrading the fungal cell walls as it tried to grow). They did statistical analysis and confirmed that all the transgenic extracts (from various lines) significantly inhibited fungal growth compared to the control extract, whereas the control extract itself had negligible effect on the fungi. This clearly demonstrated that the antifungal proteins present in the transgenic barley were biologically active and could directly suppress fungal growth and development in these in vitro scenarios. These lab assays provided compelling evidence that the concept worked: the antifungal proteins produced in the barley weren’t just sitting there, they actively hampered the fungus. The reduction in spore germination and colony growth indicated that the transgenic barley had endowed the extracts with fungal-fighting properties. This set the stage for the more realistic tests on whole plants.
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