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 1-3).
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.
Selection of shoots induction medium for callus
From previous studies, there were many studies on the ratio of hormones required for the differentiation of potato callus. Qiu [19] showed the optimum shoot induction medium of Shapody and Fovorita were MS+ ZT 2.0 mg/L+NAA 0.1 mg/L+GA3 5 mg/L and MS+6-BA 2.0 mg/L+ZT 2.0 mg/L+GA3 5 mg/L, respectively. The research of Zhang [20] indicated that the concentration of 6-BA with the highest shoot induction rate were Atlantic, Fovorita, Shapody were 2 mg/L and Zi-Huabai and Desiree were 1 mg/L. and the GA3 also had different concentration in different species. The results of others research also indicated that the hormone ratio required for different genotypes of potato to differentiate into shoots was different, and the differentiation efficiency was similarly different [21-25].
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).
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.
Optimization of agrobacterium infection conditions
Infection concentration and co-cultivation time were the transformation of the factors influence based on agrobacterium infection. Some researchers also analyzed and adjusted the agrobacterium concentration, co-culture time and infection method, and finally obtained a better way [18, 27-30]. But everyone had their own method which might be related to the type of agrobacterium and potato varieties. And a lot of studies used OD600 from 0.3 to 0.8 and Formation of callus and inhibition of agrobacterium was the key indicator of selecting. We used about 30 explants per treatment to observe there growth (Table 3).
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 co-cultivation 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 [31].The tolerance of transgenic plant cells to hygromycin depends on the copy number of the hygromycin resistance gene and its position in the nuclear genome. Plant cell death by hygromycin could produce phenolic compounds and their vacuolar constituents could negatively influence other cells. The cell death was not only because of the hygromycin concentration, but also the poisonous components produced in dead cells [32]. The suitable concentration of hygromycin was studied in potato stem, leaf and callus in our research.
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) and had same result as stem and leaf. Almost a half of callus cultured in 8 mg/L hygromycin were poorly growth and the higher concentration were led callus dead more and faster, about 15 days could be observed the dead callus (Figure 4C). And the lower concentration could not screen the positive transgenic callus and hardly being the purpose concentration (Figure 4A). It could play a screening role and not affect the growth of transformed callus under the hygromycin concentration was 8 mg/L (Figure 4B).
Combined with the results we finally selected the 8 mg/L as a selection of transgenic plants. Apparently, the numbers of survival calluses were significant reduction when hygromycin concentration was 10 mg/L, and the other concentration were no obvious difference in the screening of transformed callus. Kashani and their team showed the suitable hygromycin concentration were 7.5, 12.5 and 10 mg/L for Desiree, Agria and Marfona cultivars, respectively [33] and their results were closely similar to mine. 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-E24.2-15R vector contained hygromycin resistance gene sequence which the plants could not have these gene in nature. We used HYG primer to amplify the transgenic plants and wild DM as negative control and pFX-E24.2-15R vector as positive control to confirm the transgenic plants. The results of PCR showed the positive and transgenic plants had same strip, about 335 bp and the negative had nothing. 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.