Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 4 used is not static, it depends on which step the transformation is carried out. Taking cucumber as an example, the concentration during the early screening is generally 25 mg/L. When it comes to the rooting stage, it is necessary to add 100 mg/L to be sufficient (Chai et al., 2020). If the concentration is set too low, it may not be clean, and if it is too high, it may easily cause the regenerated tissue to be "inadvertently injured". In terms of reporter genes, GUS (β-glucuronidase) should be the most common type. It's good that it can make people intuitively see whether the transformation has been successful. It is intuitive to determine whether the gene is expressed through tissue staining, whether it is transient or stable (Thiruvengadam et al., 2013; Bhatt et al., 2021). Therefore, screening is not just a technical step, but also a "quality barrier" that must be maintained. 4 Protocols for Genetic Transformation in Cucumis 4.1 Transformation in Cucumis sativus (cucumber) Agrobacterium rhizogenes has been used in the study of gene transformation in cucumbers, especially in the production of complex plants with transgenic root systems. However, traditional methods often have many steps and are complicated to operate, so they also choose the experimental conditions. In order to save trouble, Fan et al. (2020) proposed a more direct solution. They did not use the conventional two-step method and did not set up any complicated culture medium. The method is actually quite simple: inoculate Agrobacterium rhizobium with the target gene directly into the oblique incision of the hypocotyl of the 5-day-old cucumber seedlings, and then plant the seedlings into moist sterile vermiculite, and all other steps are saved (Figure 2) (Fan et al., 2020). It sounds a bit risky, but the success rate is unexpectedly high-more than 90% of seedlings have grown roots with genetically modified ones. The efficiency and simplicity of this "one-step method" have opened up new ideas for the verification of promoter functions in cucumbers and root system research. While saving time and effort, it also avoids the damage problems caused by mid-way transplantation, which is a fast and stable alternative. Figure 2 Agrobacterium rhizogenes-mediated root transformation of cucumber achieved by one-step (Adopted from Fan et al., 2020) Image caption: a: Five days old healthy seedlings; b: The apical portion of the hypocotyl was cut diagonally (0.5 cm cut) in the liquid of K599 harboring the desired gene construct; c: A slant cut of the residual hypocotyl was scraped and inoculated on the plate grown A. rhizogenes K599 harboring the desired gene construct. Note the bacterial mass coated in the slant cut; d: Explant inoculated was directly planted into a pot with wet sterile vermiculite; e: An example of composite plant induced at 21 dpi. Bars = 1 cm (Adopted from Fan et al., 2020) 4.2 Transformation in Cucumis melo (melon) The conversion technology of Cucumis melo was not smooth from the beginning. In the early days, problems such as tetraploidy, chimera, and escape often made it difficult to advance the experiment. However, in the early 1990s, the situation began to change. Akasaka-Kenedy et al. found a breakthrough through the liquid cultured somatic
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