## 3 Factors Influencing the Genetic Transformation of Cucumber
### Bacterial Cell Density and Length of Infection  
Cucumber transformation efficiency is significantly affected by bacterial cell density and the duration of infection. [PERSON] et al. [56] investigated the effect of bacterial cell density on the transformation of unfertilized ovary and found that lower density with an optical density at 600 nm (OD\({}_{600}\)) of 0.3 was beneficial to embryogenesis, whereas higher density (OD\({}_{600}\) = 0.6) decreased infection efficiency. The study also explored the optimal infection and suitable co-cultivation times, which could help to establish a genetic transformation system for unfertilized ovarian cells. Similar results were also obtained in several different cucumber genotypes by [PERSON] et al. [54] and [PERSON] et al. [44] in cotyledons: with an increase in bacterial cell density, the _Agrobacteriumium_ grew too much, and the explants browned and died. However, other results showed that lower density was not effective, and on the basis of the plant regeneration capacity or transformation efficiency, OD\({}_{600}\) = 0.6-0.8 was recommended [18; 53]. At this time, the strain is in the logarithmic growth phase and has high infective activity. It is worth mentioning that the bacterial cell density measured by the OD value method contains both live bacteria and dead bacteria, which cannot represent the true viability of _Agrobacteriumium_; thus, the optimal bacterial density needs to be further explored [52].  
The inoculation time will be affected by selected genotype, bacterial cell density, and _Agrobacteriumium_ viability, so resistant shoots can be obtained within minutes or tens of minutes [45; 49]. However, in general, 10-20 min of inoculation time has usually been used for cucumber genetic transformation [47; 50; 57]. In addition, certain measures can be taken to promote the contact between _Agrobacteriumium_ and explants. Silwet-76 as a surfactant was added to an _Agrobacteriumium_ suspension. This effectively increases the rate of \(\beta\)-glucuronidase (GUS) staining as well as the differentiation of shoots [51]. [PERSON] and [PERSON] [33] demonstrated that explants picked with a needle underwent transient transformation more often than controls, showing that such wounding treatment was effective in improving transformation efficiency. A physical method using vacuum infiltration was developed to enhance _Agrobacteriumium_ infection by [PERSON] et al. [40]. The average transformation efficiency was as high as 11.9 \(\pm\) 3.5% in four replicate transformation experiments. Successful transformation also has been achieved by [PERSON] et al. [21]: explants were placed in a beaker with _Agrobacteriumium_ suspension and vacuum infiltrated twice for 5 min, then moved into a shaker for an additional 5 min, giving a final transformation frequency of 26%. However, the use of vacuum pumps is cumbersome, hindering the promotion of this physical method. The procedure was greatly simplified by [PERSON] et al. [49], in that the vacuum pump was replaced with a simple syringe, and green fluorescent protein detection showed that the regeneration site was not consistent with the infection site. Compared with pure immersion infection, vacuum negative pressure can significantly improve the infection depth of _Agrobacteriumium_ so that it can reach the regeneration site inside the explants [49].