Triticeae Genomics and Genetics, 2025, Vol.16, No.2, 79-91 http://cropscipublisher.com/index.php/tgg 76 Growing on and propagating: All the putative transgenic plantlets were transferred to soil in a greenhouse to grow to maturity. They were allowed to flower and set seed. The seeds (T1 generation) from each plant were collected for further molecular analysis and for planting the next generation. Throughout this process, they also maintained control (non-transformed) embryo cultures to ensure that the tissue culture process itself wasn’t causing any unexpected changes. No transgenic plants were found in the control (which is good, meaning no contamination or accidental escapes). The researchers also noted an alternative transformation method: biolistic bombardment (also known as the “gene gun”). This method involves literally shooting tiny DNA-coated metal particles into plant cells or tissues. While they primarily used Agrobacterium for this project (because it tends to produce more predictable single-copy integrations and less tissue damage), the gene gun method can be useful for barley varieties that are hard to transform using Agrobacterium. Both Agrobacterium-mediated transformation and biolistics have been successfully used to produce transgenic barley that can grow, flower, and set seed normally (Ritala et al., 2004; Gao et al., 2024). 3.3 PCR and southern blot for transgene confirmation After obtaining the regenerated plants, the next step was to confirm which ones truly had the antifungal genes integrated into their genome. The team first did a quick DNA-level screen using PCR (polymerase chain reaction). They extracted genomic DNA from young leaves of each putative transgenic plant (using a rapid DNA extraction method suited for screening). Then, using primers specific to the Chi and AMP genes, they ran PCR on each sample (Viktorová et al., 2019). If a plant was truly transgenic, the PCR would amplify a DNA band of the expected size corresponding to the inserted gene. Indeed, most of the healthy plants showed a clear band for the transgene, indicating they were positive. Any plant that didn’t show the band was considered a false positive (perhaps a plant that survived the selection due to an escape or some other reason) and was discarded. With PCR-positive plants identified, the researchers then performed a Southern blot analysis on a subset of those lines to get more information about the transgene integration. They selected ten representative transgenic lines and extracted high-quality DNA from them. This DNA was digested with specific restriction enzymes, separated on an agarose gel, and then transferred onto a membrane. They used a probe - a labeled DNA fragment corresponding to the antifungal gene - to hybridize with the membrane. If the transgene was integrated into the plant’s genome, the probe would bind to those fragments on the membrane and show up as bands. The Southern blot results showed that most of the tested transgenic plants had one or two bands. In fact, about 60% of the lines had a single-copy insertion of the transgene, and the remainder had two copies (based on the distinct band patterns). A single-copy insertion is often preferable because it usually means a simpler genetic situation and possibly more stable expression. These findings (one or two insertions per plant, with a bias toward single copies) are consistent with what’s commonly seen with Agrobacterium-transformed barley. By this stage, the researchers had a collection of confirmed transgenic barley lines, each carrying either the chitinase gene, the AMP gene, or both (in the case of co-transformed lines), and they knew roughly how many copies of the gene were in each line. The next steps were to grow the progeny of these lines and assess how well the antifungal genes were expressed and whether they actually made the plants more disease-resistant. 4 Expression Analysis and Protein Detection 4.1 Transcript analysis by qRT-PCR First, the team checked whether the antifungal genes were actually being expressed at the mRNA level in the transgenic barley. They extracted total RNA from the leaves of the transgenic barley seedlings (and from control seedlings for comparison). To ensure the RNA samples were clean, they treated them with DNase to remove any trace of DNA (so that subsequent analysis measured only RNA transcripts, not any leftover DNA). Then they performed quantitative real-time PCR (qRT-PCR) using primers specific to the Chi and AMP transgenes. For normalization, they used barley’s own housekeeping genes (like Actin or EF-1α) as reference controls.
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