Optimization of Leaf-disk Transformation and High Frequency Regeneration of Nicotianan benthamiana

Nicotianan benthamiana is a close relative of Nicotiana Tabacum. It’s widely used in studies of “Plant-Pathogen interactions”. Leaf-disk plant transformation method is widely used in plant molecular biology. In this research, we compared different parameters of different steps of Nicotiana benthamiana leaf-disk transformation procedure. We found co-cultivation at 18°C, balances the transformation frequency and late on selection better than the commonly used 25°C. Upon this pre-condition, we optimized parameters of the other steps of leaf-disk transformation of Nicotianan benthamiana. In the meanwhile, the research optimized the concentration of AgNO3 and Phytagel of regeneration medium, in the context of bud’s induction. The research found that adding 4.3 mg/LAgNO3 and 8.8 g/L phytagel in the medium, gives the highest buds induction rate.

AgNO3 is an inhibitor of ethylene synthase ACC (Eapen and George, 1997;Nail and Chand, 2003), which is widely used in plant tissue culture to improve regeneration efficiency. Pestana (1999) used AgNO3 to induce peanut callus budding , it was also used in cowpea (Mookkan and Andy, 2014), maize (Du et al., 2008), Chinese cabbage (Du et al., 2000), strawberry (Qin et al., 2006), cucumber (Cao et al., 2001), Rushan, rape, Brassica napus, black cabbage (Eapen and George, 1997). On the other hand, the hardness of plant medium had a significant effect on plant regeneration and differentiation (Wang and Zhou, 2000;Zhai et al., 2010;Zhao et al., 2007). Our previous studies also found that using high concentration of phytagel in tissue culture of N.benthamiana, significantly increasing the frequency of regeneration.
The study optimized the conditions of leaf-disc transformation of N. benthamian. The effects of different concentrations of AgNO3 and different concentrations of phytagel (Affecting the hardness of the medium) on the regeneration of N. benthamiana were studied, respectively. Figure 1 GUS staining after co-cultivation at different temperatures Note: a~d: Gus staining at 18℃, 21℃, 25℃ and 28℃ 3 days after co-cultivation According to statistics, when the co-cultivation temperature was 18℃ and 21℃, the transformation rate was the highest, which was 47.92%; when the temperature was further increased, the transformation rate was significantly reduced (Figure 2), and the genetic transformation rate was significantly reduced at 25℃ and 28℃, which were 31.25% and 18.75%, respectively. The optimum growth temperature of Agrobacterium tumefaciens is about 28℃. However, the transformation efficiency is the highest at 18℃~25℃. After co-cultivation, it is necessary to enter the process of antimicrobial screening; the overgrowth of Agrobacterium is often one of the difficulties in the process of leaf-disc transformation. The results showed that the transformation efficiency of co-cultivation at 18℃ was similar to that at 25℃. Considering that the growth rate of Agrobacterium tumefaciens significantly slowed down at 18℃, which was more conducive to the inhibition of Agrobacterium in the following selection process, we thus chose this co-cultivation temperature, 18℃, as the basis to optimize the other related genetic transformation conditions. 1.1.2 Effects of different co-cultivation times on leaf-disc transformation efficiency Through observing GUS staining of N.benthamiana explants after 1 day, 2days and 3days of co-cultivation, we found that the longer the co-cultivation time, the better the GUS staining, and the better the number and quality of infected leaves were (Figure 3). When the co-cultivation time increased to 3 days, there were more spot staining areas and more leaves got stained (Figure 3c), which indicated that the infection rate of these explants was high; however, when the co-cultivation time was as short as 1 day, there was almost no staining ( Figure 4). Under the condition of 3 days co-cultivation at 18℃, Effects of different pre-cultivation times, 1 d, 2 d and 3 d, of N.benthamiana leaf disk transformation rates were compared. Each treatment had 10~16 leaf discs and was repeated three times.
As the results showed, the staining effect was better when the pre-cultivation times were 1 day and 2 days, but the staining quantity was relatively less in the later (Figure 5b, Figure 6); when the pre-cultivation time was 3 days, the staining quantity was significantly reduced (Figure 5c, Figure 6).

Effects of different concentrations of agrobacterium on transformation rate
After optimized the co-cultivation temperature, co-cultivation time and pre-cultivation time, we studied the effect of different concentrations of agrobacterium on the transformation rate. Agrobacterium tumefaciens GV3101-pCAMBIA1305.1 was cultured at 28℃ to OD600≈0.5, spin at 5 000 r/min to pellet bacterium, afterwards, re-suspend with sterilized liquid MS medium to OD600≈0.3, OD600≈0.6, OD600≈1.0, respectively, for ready infection. The leaf discs were soaked in the ready bacterium solutions for 5~7 minutes, and then send to co-cultivation at 18℃ for 3 days. 16 leaves were randomly selected for GUS staining and these results were repeated for three times.
Staining results indicated that, when OD600=0.321, the leaves were well stained, but most of them were dot blue spots, most of the leaves gave positive staining ( Figure 7a); when OD600=0.639 and OD600=1.043, the staining of leaves was obviously better, and some leaves showed larger staining area, however, there was no significant difference between the two treatments ( Figure 7b; Figure 7c).

Effects of AgNO3 concentration and phytagel concentration on induced buds' differentiation 1.2.1 AgNO3 benefits bud induction in N. benthamiana
The results indicate certain concentration of AgNO3 benefits N.benthamiana buds induction as well as regeneration. When the concentration of AgNO3 was 1.5 mg/L~4.5 mg/L, the buds was healthier than the control treatment, while the browning of callus was obvious at 6 mg/L. Nonetheless, 4.5 mg/L AgNO3 gives best results in the terms of buds' induction ( Figure 8; Figure 9).

High concentrations of phytagel benefits buds induction of N.benthamiana
In this experiment, we found that, when the phytagel concentration is of 5 g/L (Figure 10a), the medium is relatively soft and the surface water is relatively more. The callus was trapped in the medium with heavy water content, and the bud induction rate was lower. With the increase of the phytagel concentration, the number of callus increased significantly. When the phytagel concentration reaches 9 g/L (Figure 10c), the medium is harder, while callus can still be attached to the medium surface. Water contents of the callus is obviously reduced, and the bud differentiation rate is much higher. When the phytagel concentration reaches 10 g/L (Figure 10d), the medium is too hard to adhere the callus, and thus cannot be used in practical. Buds induction rates were indicated in Figure 11. In the single factor experiment, the highest buds induction rate was reached when using 4.5 mg/L AgNO3 and 9 g/L phytagel, respectively. Thus, in this design as indicated in Table 1, we took 4.5 mg/L AgNO3 and 9 g/L phytagel as the combination center, followed by employing Design Expert10 software to select CCD model and conduct two factor response surface design experiment.
According to the CCD model in Design Expert 10 software, experimental groups was designed, with 10 bottles in each group and 6 pieces of calli in each bottle (Table 2).  In order to validate of the regression equation and further confirm the influence of various factors on tobacco budding rate, the regression model was analyzed by variance (Table 3).
Data told that the regression model is extremely significant (p<0.000 1), indicating that the establishment of the model is meaningful; the mismatch term p=0.240 4>0.05, the difference is not significant, indicating that the model fitting degree is good. The composite correlation coefficient r 2 =0.968 4 and the correction coefficient of determination (Radj 2 ) were 0.945 8, which indicate that there was a good fit between the actual value and the theoretical value of N.benthamiana buds induction rate, and the regression model could be used to analyze and predict the buds induction effect of N.benthamian (Table 3). The interaction between different concentrations of AgNO3 and phytagel was analyzed ( Figure 12). Within the detection range, the response surface graph was a convex surface with the opening downward, which indicated that there was a maximum of the buds induction rate of N.benthamiana. From the contour line of 2 vs 2 interactions, it is clear that the interaction between two factors of AgNO3 concentration and phytagel concentration has significant effect on buds induction of N.benthamiana. Through Design Expert10 software analysis, the optimal parameters of N.benthamiana buds induction were 4.33 mg/L AgNO3 combined with 8.8 g/L phytagel. Under this condition, the theoretical value of buds induction rate of N.benthamiana was 87.58%. Figure 12 Response surface diagram of the interaction of two and two factors to tobacco budding rate Note: Response surface and contour map of AgNO3 concentration and phytagel concentration with respect to buds induction rate

Verification test
According to results of above response surface experiment, we design the following verification experiments. Four groups of experiments were performed, with 5 bottles in each group and 6 pieces of calli in each bottle. After two weeks, bud's induction rate of each group was counted and calculated. The buds induction rate=the number of budding calli/the total number of calli * 100% (Table 4). According to the results of three parallel experiments, the buds induction rate histogram of each group was drawn ( Figure 13).The verified buds induction rate of combination of 4.3 mg/L AgNO3 and 8.8 g/L phytagel is 85.19%, which is very close to the theoretical value 87.58% ( Figure 13). Thus, through the response surface test, we optimized the best condition of the two factors combination. Figure 13 Germination rate of each group Note: Sprouting rate of tobacco treated with four concentrations of AgNO3 and gellan gum

The optimized process and parameters of N.benthamiana leaf disc transformation method
Based on the results of the above experiments, the optimized brief process and parameters of N.benthamiana leaf disc transformation are as follows: 1) Three days in ahead of time, Agrobacterium tumefaciens was cultured by shaking at 28℃.
2) On the first day, healthy leaves of 40~60 days (After seed germination) sterile plants were selected and cut into leaf discs of~0.8cm×0.8cm, and pre-cultivated for 1day at 25℃ on MS solid medium; meanwhile, Agrobacterium tumefaciens was sub-cultured.
3) On the second day, the agrobacterium culture is grown to OD600≈0.5; the bacteria are then collected at 4℃, 5 000 r/min for 15min; and then the pellet is resuspended on MS liquid medium to OD600≈1. The pre-cultivated leaves discs were immersed in the bacterium suspension for 3-5 minutes, the excess bacterial solution was sucked out with sterile filter paper. 4) Resulted leaf discs were placed on MS solid medium containing acetosyringone (50 µM~100 µM) for co-cultivation at 18℃ for 3 days; 5) After co-cultivation, the leaf discs were rinsed 2~3 times with sterile water (containing 500 mg/L Cefalothin Sodium), and then the leaf discs were transferred to selection medium (containing 4.3 mg/L AgNO3 and 8.8 g/L phytagel with appropriate selection agents).
6) Sub-culture the leaf discs on selection medium every 2 weeks until resistant buds were obtained.
7) The resistant buds were transferred to MS0 rooting medium to form roots; afterwards, the resulted plants were then transferred to soil.

Discussion
Leaf disc method is the widely used method for the genetic transformation of tobacco. This process is affected by many factors. In order to establish an efficient leaf disc transformation system of N.benthamiana, we analyzed and optimized different affecting factors and obtained better parameters for the process.
The leaf disc transformation method is usually consisted of several steps, namely, pre-cultivation, co-cultivation, selection and the later on regeneration. Among them, inhibition of Agrobacterium growth is an important issue in selection medium. We found that lower temperature, 18℃, is as efficient as higher temperature, 25℃, for co-cultivation step. Both temperatures give good transient transformation frequency. Considering the slow growth of Agrobacterium at lower temperature, it is obviously conducive to the next selection step. On the premise of pre-cultivation at 18℃, we optimized the other parameters of the whole process. We found, the best pre-cultivation time is for one day, the best co-cultivation time is for 3 days. Under the above conditions, the best agrobacterium concentration for transformation is at OD600≈1.
Studies have shown that AgNO3 not only promotes organogenesis, but also promotes somatic embryogenesis (Zhang et al., 1997). When the concentration of AgNO3 was 1.0~5.0 mg/L, it could promote the differentiation of tobacco regeneration buds (Wang et al., 2006). We studied the effects of four concentrations of AgNO3 on the buds induction of N.benthamiana. The results showed that when the concentration of AgNO3 was 4.5 mg/L, the buds induction rate was as high as 68.06%. One the other hand, the hardness of medium had a significant effect on the regeneration of many plants. We found that when the phytagel concentration of the regeneration medium was 9 g/L, regeneration rate of N.benthamiana is around 77.78%.
Since AgNO3 alone or high concentration of phytagel itself, both can significantly increase the regeneration frequency of N.benthamiana. We hypothesized that combination use of the two may have a better effect. According to results of the single factor experiments, we used the 4.5 mg/L AgNO3 and 9 g/L phytagel as the center combination, employing CCD to design the response surface and carried out the experiments. We found that the combination of 4.3 mg/L AgNO3 and 8.8 g/L plant gel was the best combination in theory. Finally, we confirmed the theoretical values with experiments. In the end, we summarized our optimized leaf-disc transformation process of N.benthamiana.

Materials
The MS medium is lab made according classical references. Both 6-BA and NAA are from Hefei Bomei biological company; AgNO3 is from Suzhou Tian Ke company, and the phytagel is produced by EKEAR Biotech Corp, Shanghai. The N.benthamiana seeds Agrobacterium tumefaciens GV3101 containing plasmid vector pCambia1305.1 was from stock of our own laboratory.
In the experiment, different co-cultivation temperatures was tested, namely they are 18℃, 21℃, 25℃ and 28℃; Test co-cultivation time was for 1 day, 2 days and 3 days, with 14~16 explants in each group, three replicates; the pre-cultivation time was for 0day, 1day, 2days and 3days; 16 explants in each group, and were repeated three times; Agrobacterium tumefaciens GV3101-pCAMBIA1305.1 was cultured in shaker to OD600≈0.5, after centrifugation, re-suspended in liquid MS medium, to OD600≈0.3. Leaves after pre-cultivation were immersed in the bacterial solution for 5~7 minutes, then the excess liquid drained off with filter paper, and then transferred to co-cultivation medium. Eventually, the co-cultivated leaf-discs were placed on the medium containing Cef r . 400 mg/L for bacterium resistance experiment.

Optimization of concentrations of AgNO3 and Phytagel
Using MS (1 mg/L 6-BA+0.1 mg/L NAA+0.3% sucrose+5 g/L phytagel) as the basic medium, the true leaves of sterile N.benthamiana plants, of 50 days after germination, were cut into 5 mm×5 mm leaves and pre-cultivated for 3 days at 20℃. The leaves were sub-cultured on the medium containing MS (1 mg/L 6-BA+0.1 mg/L NAA+0.3% sucrose+5 g/L phytagel) to induce callus and subculture every 2 weeks.
The concentration of AgNO3 was 0, 1.5 mg/L, 3 mg/L, 4 mg/L and 6 mg/L. The phytagel concentration was 0.5%, 0.7%, 0.9% and 1% respectively. There were 6 pieces of callus in each bottle, 4 bottles in each group, and repeated three times.
Optimized AgNO3 and phytagel concentrations were chosen, the software Design expert 10 was used to design the test plan of CCD (Central composite design) model, and the corresponding response value was obtained according to the test plan, and the regression analysis of the two factors was done by fitting the response surface multiple regression, and the theoretical value of optimized tobacco regeneration frequency was obtained, and the verification test was carried out accordingly.