## 3 Factors Influencing the Genetic Transformation of Cucumber
### Selection Markers  
When explants are inoculated with _Agrobacterium_, only the cells near the incision are infected easily. In the absence of selection pressure, some untransformed cells might also initiate differentiation. Therefore, antibiotics are often added to the differentiation medium as selection agents to kill or inhibit untransformed cells. Kanamycin, hygromycin, and phosphinothricin are the most widely employed for cucumber. As the tolerance of explants to antibiotics depends on genotype and culture stage, before transformation,the optimal concentrations for shoot and root induction have been screened by adding different concentrations of antibiotics into the explant culture medium [8,45]. Normally, 50-150 mg/L kanamycin, 2-25 mg/L hygromycin, and 2-6 mg/L phosphinothicrin are applied for cucumber transformation [51]. At an initial stage of induction, the metabolism of transformed cells is not yet fully normalized, so they are easily killed by high concentrations of antibiotics before budding. Therefore, a more effective way to conduct positive screening is by establishing a concentration gradient over time. At the beginning of differentiation, a low concentration of antibiotics is added, and when the shoot primordium is fully differentiated, a higher concentration is employed to eliminate false-positive plants [9,28,33]. To inhibit _Agrobacteriumium_ contamination, bacteriostatic agents are often used in the selection medium. Apart from effectively inhibiting the growth of _Agrobacteriumium_, antibiotics should also be chosen so as not to affect plant regeneration as much as possible [23]. According to the bacteriostatic effect, contamination rate, and germination rate, 300-500 mg/L cefotaxime sodium (Cef), carbenicillin (Carb), and timentin (Tim) were applied [29,30,53]. Tim has the best antibacterial effect and the longest duration relative to Cef and Carb, but it also has the disadvantage of high cost [7]. It is inevitable that the growth of plants and the regeneration of buds will be affected by antibiotics. Therefore, non-resistance marker genes such as green fluorescent protein (GFP) and GUS have been applied to the genetic transformation of cucumber. The transient expression rate of GUS is often used to select the best conditions for infection [51], pre-culture [30], and co-culture [33]. Unfortunately, detection with a GUS assay was destructive, and identification of stable transformation events took at least 2-4 weeks [33]. In contrast, when the _GFP_ gene was used, the labeled proteins could be observed in vivo by fluorescence microscopy. The early detection of GFP expression has greatly helped in the rapid discrimination of transformants, escapes, and chimeras [6]. [PERSON] et al. [49] observed faint fluorescence at 3 days post-infection under a stereoscopic fluorescence microscope. In addition, non-antibiotic selection markers have been used in cucumber transgenesis. [PERSON] et al. [61] reported a mannose selection system, in which mannose can only be utilized as a carbon source by cucumber cells possessing phosphomannose isomerase, finding that there were few false positives and the highest transformation efficiency when the concentrations of mannose and sucrose in the medium were both 10 g/L.