Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 75 sequence was added to its 5’ end so that the chitinase enzyme would be secreted into the spaces outside the cell (where it could encounter fungal pathogens). For the AMP gene, they fused it to a signal peptide that directs proteins to the endoplasmic reticulum, and they drove it with the rice Actin promoter to ensure the antimicrobial peptide would be abundantly produced and secreted outside the cells. Each plasmid vector also carried a bar gene (which confers resistance to the herbicide glufosinate) as a selectable marker. This marker gene allowed the researchers to easily identify which barley cells had successfully taken up the transgene - only those cells could survive the herbicide. After assembling the vectors, they verified everything by sequencing, confirming that the antifungal genes were inserted in the correct reading frame with the proper promoter and terminator sequences. The final binary plasmids were named pUbi::Chi (for the chitinase construct) and pAct::AMP (for the AMP construct). In some experiments, the team even built a combined (co-expression) vector where they linked the Chi and AMP genes together in one plasmid. The idea was to have one transgenic plant express both antifungal proteins simultaneously, potentially giving it an even stronger defense (two antifungal weapons instead of one). Once all the vectors were ready, the recombinant plasmids were transferred into Agrobacterium tumefaciens (strain EHA105) using a freeze-thaw transformation method. These Agrobacterium cells carrying our antifungal genes would be the vehicles to deliver the genes into barley in the next step. 3.2 Genetic transformation methods Transgenic barley plants were produced primarily using the Agrobacterium-mediated transformation method, which is well-established for cereals. The researchers chose a barley variety known to respond well to tissue culture (such as the cultivar “Golden Promise”) to increase transformation efficiency. Here’s a simplified summary of their transformation procedure: Explant preparation: They started with immature barley embryos as the explants (the target tissue for gene insertion). These embryos were dissected out of barley seeds at a stage where the endosperm was just forming. The starchy endosperm tissue was removed, leaving the embryo, which can be induced to form callus (a mass of undifferentiated cells) in culture. Agrobacterium infection and co-cultivation: The embryos were soaked in a solution of Agrobacterium that contained either the pUbi::Chi or pAct::AMP plasmid. The bacterial concentration was adjusted (OD₆₀₀ ~0.6) and a compound called acetosyringone (200 µM) was added to the solution to activate the Agrobacterium’s gene transfer system. The embryos were immersed for about 5 minutes, and then they were co-cultured with the Agrobacterium on a medium in the dark at 28 °C for 3 days. During this time, the Agrobacterium would attach to the plant cells and transfer the T-DNA (which carries our antifungal gene) into the barley embryo cells. Selection and plant regeneration: After co-cultivation, the embryos were moved to a selective growth medium. This medium contained an herbicide (either a sulfonylurea or glufosinate, depending on the marker gene used) to kill any cells that did not receive the bar resistance gene, and an antibiotic (e.g., cefotaxime) to kill off any remaining Agrobacterium. Over the next 6-8 weeks, the plant cells that had taken up our gene formed callus and then began regenerating into plantlets. One could observe green shoots emerging from the calli. These shoots (putative transgenic barley plants) were then transferred to a hormone-free medium to encourage them to grow roots and develop into small plantlets. Initial screening: The tiny plantlets that grew were subjected to the herbicide selection (by spraying or adding the herbicide) to double-check that they carried the resistance gene - those that survived were considered likely transgenic plants. On average, the team was able to regenerate about 3-5 transgenic plants out of every 100 embryos they started with, which is comparable to previously reported transformation efficiencies for barley. When they used the larger co-expression vector carrying both genes, the transformation efficiency was a bit lower (likely because the plasmid was bigger and more complex), but they still obtained a number of dual-gene transgenic plants.
RkJQdWJsaXNoZXIy MjQ4ODYzNA==