Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 2 2 Agrobacterium-Mediated Transformation: Basic Principles 2.1 Mechanism of T-DNA transfer Agrobacterium rhizogenes are considered an old acquaintance when sending foreign genes into plants. This transformation method is widely used mainly by a bacteria called Agrobacterium tumefaciens. It is not a high-tech product, but a bacteria in nature that can "secretly" stuff a piece of DNA (called T-DNA) into the plant genome. This T-DNA stays on its Ti plasmid, usually quiet, but once the bacteria hit the wound part of the plant, things start to work (Lacroix and Citovsky, 2013). How did T-DNA be transferred ? There are still many steps. At the beginning, plant wounds will release some phenolic compounds, such as ferulic acid ketone, which trigger the vir gene activation in the bacteria. Then, the bacteria cut the T-DNA off the plasmid and then wrapped it with proteins such as VirD2 and VirE2 to form a structure called the T complex (Gelvin, 2010). This complex will then enter the plant cells and then enter the nucleus. However, entering the nucleus is not the end point. Next, the T complex will be untied, and T-DNA will then be inserted into its genome through the plant's own DNA repair mechanism. The insertion location is usually random, but the target gene can be expressed stably as long as the integration is successful (Gelvin, 2010). Of course, this whole process does not necessarily mean success. Whether bacterial strains are selected well and whether the plant cell status is good will affect the final transformation efficiency (Lacroix and Citovsky, 2013). 2.2 Factors influencing transformation efficiency When using Agrobacterium rhizogenes to make the cucumber (Cucumis sativus L.) gene transformation, efficiency is not simple, it is affected by many factors. Sometimes even if the steps are the same, the results may be completely different. Such as the genotype of cucumber, the age of the explant, the type of strain used, and even the details of the infection will determine the success or failure. There is a big difference between genotypes, and this cannot be ignored. Cucumbers in northern China perform much better in bud regeneration than pickled or sliced varieties in the United States (Figure 1) (Liu et al., 2023). Explants are more popular at younger ages, such as cotyledons or hypocotyls near the apex, and their regeneration ability is generally higher (Liu et al., 2023). Let’s talk about the strain. Not all strains are common, for example, the positive frequency of AGL1 is higher than that of GV3101, while EHA105 is more suitable for use on cucumber variety Poinsett 76 (Wang et al., 2015). So how to choose a strain is also a science. Of course, the handling conditions are also critical. What is the concentration of bacterial solution you use? Is ferulic acid ketone added? How long does infection and co-culture last? Once these variables change, the result may be different. Some people also found that precultivating explants in advance, or wiping the ingredients of the culture medium can help the formation of transgenic buds (Thiruvengadam et al., 2013). The screening stage should not be taken lightly. The use of screening agents such as kanamycin is actually quite effective. They can screen out the truly transformed cells without wasting time (Thiruvengadam et al., 2013; Liu et al., 2023). 3 Techniques for Enhancing Transformation Efficiency 3.1 Optimization of agrobacterium strains 3.1.1 Strain selection and genetic modifications Not all Agrobacterium rhizogenes are suitable for use on cucumber plants, which has become apparent in the experiment. Some strains show better than others. For example, the conversion efficiency of AGL1 is much higher than that of GV3101 (Liu et al., 2023). Although GV3101 may be OK on other crops, it is not very useful when it comes to the genus Cucumber. In addition to these two common strains, the transformation results of EHA105 and LBA4404 in some other plants are also quite good, which also shows a problem-the selection of strains cannot be casually (Thiruvengadam et al., 2013). Moreover, if the effect of ready-made strains is not satisfied, some studies simply use hands-on modification, such as introducing plasmids such as pCAMBIA2300 into the strain to improve their transformation ability (Bhatt et al., 2021). From this perspective, not only do you have to choose the right strain, but sometimes you have to "train" it.
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