Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 72 Feature Review Open Access Functional Characterization of a Transgenic Barley Line Expressing Anti-Fungal Protein Ming Li, Congbiao You Tropical Microbial Resources Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China Corresponding email: congbiao.you@hitar.org Triticeae Genomics and Genetics, 2025, Vol.16, No.2 doi: 10.5376/tgg.2025.16.0009 Received: 25 Feb., 2025 Accepted: 02 Apr., 2025 Published: 18 Apr., 2025 Copyright © 2025 Li and You, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Li M., and You C.B., 2025, Functional characterization of a transgenic barley line expressing anti-fungal protein, Triticeae Genomics and Genetics, 16(2): 79-91 (doi: 10.5376/tgg.2025.16.0009) Abstract Fungal diseases pose a serious threat to barley yield and quality, and it is difficult to breed highly resistant varieties quickly using traditional methods. Antifungal proteins, as crucial components of a plant’s innate immune system, have multiple defense functions (like breaking down cell walls, damaging membranes, and inhibiting pathogens), offering a new strategy for improving crop disease resistance. This study describes the sources and functions of antifungal genes, analyzes transgenic barley materials expressing antifungal proteins, and systematically characterizes their molecular, biochemical, and biological functions. Gene expression levels and protein accumulation in the transgenic plants were examined using qRT-PCR and Western blot, and tissue-specific expression was investigated via immunolocalization. The study also summarizes three case studies: a chitinase-producing barley from Japan’s NARO institute, an antimicrobial peptide-expressing line developed by ICRISAT, and a BDAI-overexpressing line from the University of Copenhagen. These cases further demonstrate the feasibility and promising prospects of using antifungal protein genes in transgenic strategies. The findings provide important evidence that genetic engineering can enhance barley’s disease resistance, and they offer theoretical and practical guidance for breeding disease-resistant crops. Keywords Barley; Antifungal protein; Chitinase; Disease resistance identification; Agronomic traits 1 Introduction Barley (Hordeum vulgare) is one of the most widely grown cereal crops in the world. It’s used not only for brewing and food processing but also as an important feed grain for livestock. Thanks to its broad adaptability and tolerance to drought and cold, barley can thrive in marginal environments and plays a unique role in food and feed security. In fact, global barley production has been holding steady at roughly 150 million tons per year in recent years, putting it among the top cereal crops. In China, barley is mainly grown for beer brewing and as animal feed. Even though it’s cultivated on a relatively smaller scale, it’s vital for food security and livestock farming in certain regions (like the Qinghai-Tibet Plateau). But barley farmers still face major challenges, especially from pests and diseases. Fungal diseases such as powdery mildew, leaf rust, and Fusarium head blight can drastically reduce barley yields and grain quality (Oğuz and Karakaya, 2021). That’s why boosting barley’s disease resistance has long been a key focus in crop research and breeding. Various fungal diseases threaten barley production, including Fusarium head blight, powdery mildew, net blotch, and leaf rust. These diseases lead to lower yields and poorer grain quality. For instance, Fusarium head blight (also known as scab) can shrivel the grains and contaminate them with toxins, impacting the safety of the harvest. Leaf diseases like powdery mildew and rust impair photosynthesis and reduce the thousand-grain weight of the crop. Traditional breeding for disease resistance relies mostly on finding resistance genes in wild or cultivated germplasm and crossing them into crops. However, fungal pathogens evolve so quickly that resistance genes which once worked can lose effectiveness in just a few years (Singh et al., 2019; Fernando et al., 2020). This makes it really hard to achieve long-lasting, broad-spectrum disease resistance using conventional breeding alone. Transgenic breeding provides a new approach to tackling this problem by introducing specific disease-resistance genes into barley, thereby equipping the plants with novel ways to fight infections. In particular, some antifungal proteins (such as chitinases, β-1,3-glucanases, antimicrobial peptides, and defensins) can directly inhibit the growth and development of pathogenic fungi. If these antifungal protein genes are expressed well in the plant,
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