Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 3 3.1.2 Impact of virulence factors When it comes to gene transformation, virulence factors are not optional. They can directly affect the results, especially when you want to increase conversion efficiency. Many people have tried adding ferulic ketone into the culture medium, and the effect is indeed quite obvious. Plants like Mangji and cucumber, with ferulic acid ketone when infected or co-cultured, the conversion rate is significantly higher (Chai et al., 2020; Bhatt et al., 2021). But then again, you can't do anything randomly if you add it. It is useless to have too low concentration, and it may be suppressed if it is too high. Studies have found that the transformation effect is best with 100 µM ferulic acid ketone, especially in the pre-culture and co-culture stages (Udayabhanu et al., 2022). Therefore, it is not only a question of whether to add or not, but also a fine control of how much to add and how much to add. Figure 1 Agrobacterium stain AGL1 exhibited higher transformation efficiency than GV3101 (Adopted from Liu et al., 2023) Image caption: Transgenic plants were developed with RNAi constructs for the CsAPRR2 gene in a PS76 background. Plantlets in the jars were generated from GV3101-mediated (A) and AGL1-mediated (D) transformation, respectively. T0 transgenic plants grown in soil were mediated by GV3101 (B) and AGL1 (E), respectively. PCR verification identified positive T0 transgenic plants from GV3101-mediated (C) and AGL1-mediated (F) transformation, respectively. In (C,F), the first lane is a size marker, and lanes 1-4 are the positive control and three regenerated plantlets from independent transformation events. Scale bar = 1.0 cm (Adopted from Liu et al., 2023) 3.2 Pre-culture and co-cultivation conditions The conversion into unsuccessful results are sometimes not a problem with bacteria, but a lack of good grasp of the operation details. The two stages of pre-cultivation and co-cultivation are simple and simple, and the details are quite particular. Environmental conditions, processing time, whatever is wrong, the conversion efficiency may be reduced in the end. For example, the experiment of Brazilian rubber tree found that the T-DNA transfer effect was much better when controlling the co-culture temperature at 22 °C and allowing the explant to stay in the dark for 84 hours (Udayabhanu et al., 2022). The research on cucumbers is similar. Pre-culture plus dark conditions, combined with a little ferulic acid ketone can also significantly improve the conversion effect (Chai et al., 2020). However, once the conditions are done, the materials themselves cannot be ignored. Some explants are older, but they are not very "obedient". Generally speaking, the younger the tissue, the higher the conversion rate, which has also been verified in cucumber experiments (Liu et al., 2023). 3.3 Selection markers and reporter genes How to determine whether the conversion has been successful, You have to choose the right tool. Marker genes and reporter genes come in handy at this time. Kanamycin is a commonly used selective antibiotic, but the amount
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