Molecular Microbiology Research, 2025, Vol.15, No.1, 1-9 http://microbescipublisher.com/index.php/mmr 5 embryo system and successfully solved these difficult problems (Nonaka and Ezura, 2015). Later, a research team tried to use the entire germinated seed as an explant, and regenerate the cotyledon nodule area and stem junction as targets. The effect is not bad, the conversion frequency of melon reaches 13%, and plants with exogenous genes can be detected by waiting for about a month (Zhang et al., 2014). More importantly, these transgenic plants are almost exactly the same as wild type in appearance and growth, and there are no obstacles to commercialization. Of course, the increase in regeneration rate has never stopped. Researchers have successively tested microbrushing, sonication, vacuum infiltration and other operations, with the purpose of improving infection efficiency. Some people simply start from the molecular level, such as overexpressing developmental regulatory genes such as AtGRF5 and AtPLT5, which does bring about higher conversion success rates and makes conversion less dependent varieties (Wan et al., 2023). Speaking of Oriental Melo (Cucumis melo var. makuwa), there is also a specialized method. Cotyledons were used as explants, benzyl adenine and indoleacetic acid were added to MS medium, and then GUS staining, PCR and Southern hybridization were used to confirm whether the transgene was integrated (Choi et al., 2012). When using kanamycin and genetic dyskinin during screening, the conversion rates were 2.9% and 7.1%, respectively. Although it is not too high, it has practical value. And when it comes to gene editing, researchers are not idle either. Strategies like "optimal infiltration intensity" are specially customized for cucurbita plants and optimize the transformation process. Using this method, CRISPR/Cas9 has successfully knocked out some genes from the ERECTA family and obtained mutants with shorter internodes and more compact plants (Xin et al., 2022). 5 Challenges and Solutions in Cucumis Transformation 5.1 Recalcitrance and low transformation rates When using Agrobacterium rhizogenes for gene transformation of Cucumis, an old problem has not been completely solved - it is not easy to "serve" and is not easy to transform. Especially cucumber (Cucumis sativus L.), the conversion efficiency has been low. Although technology has made a lot of progress in recent years, it is still not easy to make it stable "receive" foreign genes. There are many factors that affect efficiency, and we cannot just blame the technology for not being able to do it. Such as the genotype of cucumber, the origin and age of explants, the selection of strains, etc., they may all influence the experimental results. For example, the regeneration rate of cucumber buds in northern China is much stronger than that in the United States, which shows that the genotype will indeed widen the gap (Liu et al., 2023). There are also some considerations in explants. Organs that are too old are not very "obedient", and young tissues are generally better used. Faced with these difficulties, the researchers did not fail to do anything. These methods have been tried, such as adjusting infection and co-culture conditions, and using screening agents such as kanamycin, and have certain effects. In terms of strains, AGL1 has a higher conversion efficiency than GV3101 (Liu et al., 2023). Some people have tried to add ferulic acid ketone to the culture medium at different stages, and the results show that the conversion efficiency has also been improved (Chai et al., 2020a; 2020b). Overall, the conversion rate can reach about 0.2% to 1.7%. It is not high, but it has been a significant improvement compared to the situation that was almost impossible to achieve in the early years (Liu et al., 2023). 5.2 Somaclonal variation and genetic stability Somatic mutation is also a headache, especially during the transformation process, which may cause genetic instability in the plants. Sometimes, even though the target gene is introduced, the grown plants do not perform as expected, and the problem may lie here. This phenomenon is actually not uncommon in the tissue culture of cucumbers (Pawełkowicz et al., 2021). The mechanism of somatic cell mutation has not been fully understood yet, and it is indeed complicated to say that it is complicated. However, judging from the analysis of the transcriptome, some clues can still be found. For example, polymorphisms in gene regions may have some interaction with molecular networks, which will activate specific signaling pathways, which in turn affect gene expression (Pawełkowicz et al., 2021). Although this kind of mutation brings trouble, it is not entirely worthless - sometimes, it can provide some useful genetic differences that may come in handy in breeding.
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