Improved the Agrobacterium tumefaciens -mediated 1 transformation of cucumber by a modified the using of 2 antibiotics and acetosyringone 3

13 Background: Cucumber ( Cucumis sativus ) is one of the most important vegetable crops 14 in the world. As conventional breeding of cucumber is very challenging, genetic 15 engineering is an alternative option to introduce important traits such as enhanced stress 16 resistance and nutritional value. However, the efficiency of the transformation system 17 depends on genotypes, transformation conditions, selection agents, etc. This study aims 18 to speed up the process of Agrobacterium -mediated transformation of cucumber. ‘ Xintai 19 mici ’, a very popular and typical north China-type cucumber variety, was transformed with 20 Agrobacterium GV3101. The strain carried pCAMBIA2300s plasmid, a double vector with 21 the marker gene of neomycin phosphotransferase II ( npt II). 22 Results: The research results indicated that cefotaxime sodium was suitable for inhibiting 23 Agrobacterium in the stage of screening and bud elongation. Timentin was best used 24 during rooting stage. Furthermore, 25 mg/L kanamycin was used in the early stage of 25 screening and increased to 50 mg/L for further screening. At the bud elongation and 26 rooting stage, 75 and 100 mg/L kanamycin was used respectively to improve the 27 screening efficiency. In order to obtain the highest regeneration frequency of resistant 28 buds, 50, 150, and 100 μM acetosyringone were added in the pre-culture medium, 29 infection solution, and co-culture medium respectively. To confirm the presence of the 30 transgenes, DNA from npt II transgenic cucumber plants was analyzed by polymerase 31 chain reaction after transplanting resistant regenerated plants. 32 Conclusions: We finally achieved an 8.1% conversion, which was among the highest 33 values reported until date using cucumber ‘ Xintai mici ’. Thus an effective protocol for 34 Agrobacterium tumefaciens -mediated genetic transformation of cucumber was optimized. 35


38
Cucumber (Cucumis sativus) is one of the most important vegetable crops widely grown in 39 regeneration buds of 0 and 100 mg/L treatments were significantly higher than that of any other 112 treatments. There was no significant difference among 200, 300 and 400 mg/L treatments (Fig.  113 2-f). 114

Effects of acetosyringone on regeneration of resistant buds 115
Different concentrations of acetosyringone were added respectively to four important stages 116 of genetic transformation respectively, the pre-cultivation ( Fig. 3-a), infection ( Fig. 3-b), 117 co-cultivation (Fig. 3-c), and selective culture stages (Fig. 3-d). The regeneration frequencies of 118 resistant buds were counted respectively after 28 d and shown as follows. 119 In the test of adding acetosyringone in the pre-cultivation stage, the frequency of resistant 120 buds increased initially and then decreased with the raise of acetosyringone concentration. 50 μM 121 treatment had the highest frequency of resistant buds (53.3%) and which was significantly higher 122 7 than that of 100, 200, and 400 μM treatments. Whereas, acetosyringone greater than or equal to 123 100 μM showed severe inhibition to the frequency of resistant buds. There was no notable 124 difference between 0 and 50 μM treatments (Fig. 3-e). As shown in Fig. 3 buds of 0, 50, and 100 μM treatments were at the same difference level, and 50, 100, 150, and 200 139 μM treatments were sharing another difference level. Taken together, these results suggested that 140 extra acetosyringone could increase the regeneration frequency of resistant buds while in the early 141 stages of transformation, but not after it was been screening (Fig. 3). 142 8

Polymerase chain reaction (PCR) analysis 143
The regenerated plants of cucumber were domesticated. And the total DNA of the ninth 144 tender leaf was extracted. The primers of report gene npt II were used to identify the transformed 145 plants ( Fig. 4-a), and a 480 bp product was amplified, which was the same as the positive control. 146 The primers of the Agrobacterium genome were used to eliminate the contamination of plants 147 from Agrobacterium, and the total DNA of regenerated plants was not amplified except for lane 12, 148 17, and 18 ( Fig. 4-b). DNA of wide-type plant and negative control were not amplified (Fig. 4). 149

150
This study was conducted to establish an Agrobacterium tumefaciens-mediated 151 transformation system for cucumber. We evaluated the optimal dosage of kanamycin and various 152 antimicrobial antibiotics by observing the growth of explants and Agrobacterium in different 153 concentrations. The addition amount of acetosyringone was changed when applied to different 154 culture stages. Then the optimized regeneration protocol was adapted to transformation for 155 cucumber. 156

Effects of kanamycin on explants 157
Kanamycin has a great inhibitory effect on untransformed explants, especially the growth of 158 root [25]. Therefore, kanamycin was widely used as a selection marker with successful results [26, 159 27]. The screening concentration of different cucumber varieties needed to be explored owing to 160 the different sensitivity to kanamycin. As shown in Fig. 1, the regeneration of the buds was 161 inhibited completely at 75 mg/L kanamycin, but the explants began to brown at the same time. In 162 9 order not to affect the regeneration of the delicate explants, 25 mg/L kanamycin was used after 163 co-culture and then raised to 50 mg/L for further screening without browning. In addition, the 164 concentration of kanamycin could be increased to 75 and 100 mg/L to prevent the emergence of

Effects of kanamycin and bacteriostatic antibiotics on Agrobacterium and explants 271
Explants that were not being exposed to Agrobacterium were placed on the medium-II. PCR amplification was performed as follows: 94℃ for 3 min, 35 cycles of 94℃ for 30 s, 58℃ for 315 30 s, 72℃ for 1min, followed by a final extension of 72℃ for 10 min. PCR products were 316 separated on a 1% agarose gel and visualized by ethidium bromide staining. 317 Ethics approval and consent to participate 318 Not applicable. 319

Consent for publication 320
All authors agreed to publish this manuscript. 321