Construction of Potato DM1-3-516-R44 (DM) Transgenic System Based on Agrobacterium Transformation

Background: The sequencing potato DM1-3-516-R44 played an irreplaceable role in the study of gene function. So far, no one research the transformation system about DM. Therefore, our experiment was studied from three aspects: plant regeneration system, optimization of agrobacterium infection conditions and the effect of hygromycin on DM. Results: A relatively suitable method for genetic transformation of DM was obtained: 1) The stem callus induction medium was MS + IAA 0.5 mg/L + 6-BA 2.0 mg/L, the leaf callus induction medium was MS + NAA 1.0mg/L + 6-BA 0.5mg/L and the shoot differentiation medium was MS + 6-BA 3.0 mg/L + ZT 0.5 mg/L. 2) The specific transformation condition was the agrobacterium concentration kept the OD600 = 0.3, and co-culture time consisted 3 days in the dark. The hygromycin concentration chose 8 mg/L to screen the transgenic plants. 3) Using hygromycin to screen about 100 transgenic shoots, 75 shoots were obtained and 53 strains were identified had target stripe by PCR technology. Conclusion: The efficiency of the transformation system we created was over 50%. It provided a good basis for the study of potato gene function.

genetically modified crops of corn, soybean and cotton which contained insect and herbicide resistant genes were mainly genetically modified crop in currently. Moreover, amylose-free line transgenic plants had also been approved for the commercialization [4]. Through gene transformation technology could possible obtain some characters that were not available in traditional breeding and enrich their genetic information. The advantage of gene transformation technology was that it did not influence the unique genetic integrity in a particular plant and many traits that were difficult to acquire from wild species could be incorporated into the genome [5, 6]. There were a variety of methods to improve crops according to genetic transformation. The two commonly methods were particle bombardment and Agrobacterium-mediated. Comparing these two methods, Agrobacterium-mediated methods had higher transformation frequency and efficiency and particle bombardment were beneficial to transform multiple genes. Although each method had its own advantages and disadvantages, the Agrobacterium-mediated method was currently the most widely used and preferred potato transformation method.
Dale was researched that only a half of 34 potatoes could be transformed with tubers and some varieties could neither be transformed with tubers nor leaves [7]. Han and their team showed that expect potato Jowon had high transformation efficiency and other species (Namseo, Chuback, Jopoong and Jasim) had lower efficiency with stems and leaves [8]. Some researchers from China [9-12] were identified some potato species but no one researched the regeneration system of sequenced species and these species had clearly genetic information to support the study of potato gene function.
Thus, in this study, a double haploid potato DM1-3-516-R44 (DM) [13] that was potato sequencing variety was used as the research object, and potato stems and leaves were used to establish an efficient regeneration system. On the other hand, optimizing agrobacterium transformation to improve transformation efficiency and provided support for research on revealing potato gene function.

Selection of callus induction medium for stems and leaves
Dedifferentiation and redifferentiation of explants was the main point of plant transformation. However, this point was based on the plant hormone ratio. Referenced some researchers results[14-18], we selected 54 combinations of 4 phytohormones to determine the ratio of hormones which were most suitable for dedifferentiation and form callus of DM (Supplement table 1 The stems could induce the calluses under the MS with 6-BA and IAA cultured for about 20 days. But different concentration could lead to different types which were the callus took root and could not differentiate when the 6-BA concentration was lower than the IAA concentration ( Figure 1A), and it was easy to produce white villi-like dense callus for a long time when the difference between 6-BA and IAA concentration was too large ( Figure   1B).
Comparing with callus types under the MS with 6-BA and NAA showed that the leaves would be brown even dead under higher 6-BA concentration ( Figure 1C) and callus took root under higher NAA concentration ( Figure 1D). Only 6-BA and NAA had equally concentration could induce healthy callus ( Figure 1E). And the combination of this hormone was not suitable for callus formation of stem.
Most of the concentration ratios could induce the loose and transparent callus or take root in MS medium containing 2,4-D of stems and leaves. Therefore, it was concluded that 2,4-D was not suitable for induce callus of DM ( Figure 1F and G).
The results showed that the leaves could not induce the healthy calluses even turn into brown and death, but the stem could form better callus and green swollen appeared at both ends under the 6-BA and IAA hormone ratio ( Figure 1H). On the contrary, under 6-BA and NAA hormone ratio, only leaves could induce better callus ( Figure 1E). Even though stems and leaves could form some calluses, but the calluses were turned brown and dead under 6-BA and 2,4-D hormone ratio. Due to these ratios could induce some stems and leaves to form calluses, thus, so we selected some hormone ratio repeated were still unsatisfactory.
Finally, we could only select 2 different hormone ratios to induce stem and leaf to form callus, respectively. We chose 2.5 mg/L 6-BA and 0.5 mg/L IAA to induce stem to form callus and 1 mg/L 6-BA and 1 mg/L NAA to induce leaf. Similar to callus induction medium selection, hormone selection also played a vital role in shoots induction. We also selected 4 hormones to determine the ratio of hormones (Supplement table 4-6) [23, 26-28]. Large difference in induction rate led to different concentration ratios. The higher the cytokinin concentration, the better the callus differentiation rate, but when the 6-BA concentration was higher than 4 mg/L, it would affect the time required for differentiation. And the NAA-added differentiation medium was not suitable for the differentiation of leaf callus. With the ratio of 6-BA and ZT, not only the differentiation time was reduced, but also the callus of leaves and stems could be affected (Figure 2A and B). Similarly, The ratio of 6-BA to GA3 was favorable for the differentiation ( Figure 2C).

Selection of shoots induction medium for callus
The results showed the shoot induce ratio was highest under 3 mg/L 6-BA with the combination of different hormone types. Almost all of callus could be induced under the hormone ZT and 6-BA (3 mg/L 6-BA and 0.5 mg/L ZT, 3 mg/L 6-BA and 1 mg/L ZT) and GA3 and 6-BA (2 mg/L 6-BA and 0.1 mg/L GA3, 3 mg/L 6-BA and 0.1 mg/L GA3). Finally, we selected 3 mg/L 6-BA and 0.5 mg/L ZT as the shoot induction because the higher inductivity and cost-effective.  Table 3).

Optimization of agrobacterium infection conditions
The results showed co-culture 2 days was not efficient for agrobacterium infection under the OD600 from 0.3 to 0.5. About half of explants were turned to tawny and others did not have obvious callus formation after 2 weeks later cultured in callus induction medium ( Figure 3C and D). Until the co-culture was extended to 3 days, a majority of explants were induced into callus ( Figure 3E and F). But the higher concentration was hard to inhibit and led the explants were dead. This phenomenon was appeared when the OD600 was 0.5 ( Figure 3A and B). To avoid this situation, we finally selected OD600 = 0.3 as infection concentration and 3 days for co-cultivation ( Figure 3

E and F).
At present, the genetic transformation method using agrobacterium as a vector had been applied to a large number of crops, especially in dicotyledons [109]. However, because infection involved the regulation of multiple factors, the transformation efficiency was not high. This experiment combined the characteristics of potato material and the optimization of the agrobacterium transformation system by Shi Hu [98], and performed preliminary optimization from the aspects of agrobacterium concentration and cocultivation time, and antibiotic screening. It was found that when potato explants were infected with a high concentration of agrobacterium, it was extremely difficult to inhibit the growth of bacteria, resulting in death of potato explants. Therefore, the concentration of agrobacterium used in this experiment was similar or slightly lower than that used by other researchers [110,111]. The screening results of co-cultivation time of callus in the dark showed that at a suitable concentration of agrobacterium, co-culture for more than 3 days would produce more agrobacterium, which was not easy to inhibit and not conducive to callus survival.

The influence of hygromycin concentration on plant
Hygromycin could prevent protein production in plant cells in the translation process The studies showed the leaves and stems were cultured in MS with Hyg for 20 days, the higher explants death and the higher hygromycin concentration (Table 4). The stems and leaves had same tendency. The higher hygromycin could led the explants all dead in about 20 days, the leaves and stems changed from green to tan, and the lower concentration could not play a role in selecting and could not even restrict explants growth, effectively.
Thus, the results indicated that 4 mg/L hygromycin could not restrict the explants growth and 12 mg/L hygromycin led the explants were all dead in a few days. And a half of explants were dead and others were turned brown till died a few days later under the hygromycin was 8 mg/L. These concentrations not only partially limited explants survival, but also certainly influenced plant growth.
We also used callus to select the hygromycin concentration (Table 5)  Simultaneously, it also verified that there were some differences in the tolerance to hygromycin between different varieties.

Identification of transgenic plants
We selected pFX-E24.2-15R vector to build the enhancer trap vector. Figure 5A showed the callus formation, differentiation and strike root. We selected some plants which had normally rooting and good growth potential for identification from a molecular way.
In order to obtain the real transgenic plants, the PCR tested the DNA could distinguish the positive plants (Fig 5B). Due to pFX-E24. The agarose gel electrophoresis showed that there was another stripe except the target one which might be the primer dimer. We finally identified 30 transgenic plants which all had brighter target stripe.

Conclusion
There had been many reports on methods for transforming potatoes based on agrobacterium infection. But the differences among varieties could had different responses of plant hormones. Combining the importance of sequenced potato DM in scientific research, we constructed a more efficient potato transformation system.
2) The specific transformation condition was the agrobacterium concentration kept the OD600 = 0.3, and co-culture time consisted 3 days in the dark. The hygromycin concentration chose 8 mg/L to screen the transgenic plants.
3) Using hygromycin to screen about 100 transgenic shoots, 75 shoots were obtained and 53 strains were identified had target stripe by PCR technology. Thus, the efficiency of the transformation system we created was over 50%.

Callus induction from stems and leaves
Referenced the results of potato regeneration system from some researchers showed the concentration and ratios of cytokinin and auxin were the main influence in callus formation process. Thus, we set the concentration of 6-BA as 0 mg/L, 1 mg/L, 1.5 mg/L, 2.5 mg/L, 3.5 mg/L and 4.5 mg/L and combined with IAA, NAA and 2,4-D at concentrations of 0.5 mg/L, 1 mg/L and 2 mg/L (Table 1) to find the best hormone ratio in callus induction medium (CIM). Statistics of callus formation cultured about 15 days after.

Induction of callus differentiation shoots
Combining with others' research, 6-BA, NAA, GA3 and ZT were used to induce the buds formation. Thus, different ratios of 6-BA and ZT, 6-BA and NAA, and 6-BA and GA3 were selected to determine shoot induction media (SIM). We finally set the phytohormone combination were list in table 2. The callus were cultured in the medium (MS) with different concentrations of hormones for 20 days to statistic the shoots induction.

Optimization of Agrobacterium infection conditions
We selected pFX-E24.2-15R (Enhancer trap) transformed Agrobacterium EHA105 to infect the potato stems and leaves. We set 4 different concentration of Agrobacterium solution, OD 600 were 0.3, 0.4 and 0.5, and co-cultivation with explants for 2 or 3 days. And the specific steps for Agrobacterium transformation were detailed below.

1.
Set an overnight culture of Agrobacterium in YEB medium with the correct antibiotics (Rif + Kan) and let grow the culture of Agrobacteria until OD 600 = 0.3, 0.4 and 0.5.
Resuspended in the same volume YEB medium without antibiotics to an OD 600 = 0.3, 0.4 and 0.5.

2.
Cut potato stems without growing point and leaves through the middle-rip one or two-fold without petiole pre-cultured in callus induction medium (pre-CIM) for about 3 days, and place them in a dish containing Agrobacterium and shake culture for 10 min.

3.
Filter paper was blotted dry, and the stems and leaves was incubated in callus induction medium (CIM) without antibiotics for 2 or 3 days in the dark.

4.
Rinsed the cultured callus with sterile water containing 500 mg/L Cef and sterile water for 4 times. Blotted dry sterile water with filter paper and transferred into CIM with 300 mg/L Cef and Hyg.

5.
After 15 days transferred the callus onto shoot induction media (SIM) and changed the media every 10 days until shoots were formed.

6.
The shoots reached a length of about 1-2 cm and transferred them into MS2 (MS with

Screening of hygromycin concentration
To avoid potato explant was all dead under the higher antibiotic concentration or the low concentration could not preliminary screening of transformed callus. Thus, we set 6 gradients, 0 mg/L, 4 mg/L, 8 mg/L, 12 mg/L, 16 mg/L and 20 mg/L to find out an optimal concentration before potato genetic transformation.
We cultivated potato stems and leaves in a plate containing MS medium with hygromycin for about 2 weeks to statistic the statement of these tissues. Moreover, we transferred the co-cultured callus into a differentiation medium containing 5 hygromycin concentration gradients, 2 mg/L, 4 mg/L, 6 mg/L, 8 mg/L and 10 mg/L. statistic of callus survival about 2 weeks later.

Identification of transgenic plants
The shoots were cut into MS medium with 300 mg/L Cef and Hyg, and cultured four weeks Tables Table 1 Hormonal