Synergistic Effect of NaCl Pretreatment and PVP on Browning Suppression and Callus Induction from Petal Explants of Paeonia Lactiflora Pall. ‘Festival Maxima’

Browning is prevalent in tissue cultures of Paeonia lactiflora Pall. (herbaceous peony), and severely affects and restricts the growth and differentiation of the explants. In this study, dipping excised explants in a sodium chloride (NaCl) solution as a pretreatment, adding polyvinyl pyrrolidone (PVP) to the culture medium, storing planted explants at 4 °C for 24 h, and transferring planted explants to a new medium after 24 h were considered as browning-suppression methods in tissue cultures of herbaceous peony ‘Festival Maxima’. The treated petal explants were cultured in a culture room with a 16-hour photoperiod, 25 °C temperature, and 80% relative humidity in darkness for 4 to 8 weeks. The results demonstrated that dipping excised explants in a 0.5 g·L−1 NaCl solution, adding 0.5 g·L−1 PVP to the medium, storing planted explants at 4 °C for 24 h, and transferring planted explants to the same fresh medium after 24 h could effectively inhibit browning. Adding PVP to the medium led to the greatest browning suppression percentage of 95%. Storing planted explants at 4 °C for 24 h reduced the effectiveness of other treatments in suppressing browning. After 8 weeks, dipping excised explants in a NaCl solution resulted in the highest callus induction percentage of 75%, while storing explants at 4 °C for 24 h suppressed callus formation. It was observed in all treatments that decreases in browning was accompanied with higher levels of phenols and lower activities of phenylalanine ammonia-lyase (PAL) and polyphenoloxidase (PPO). Overall, the results suggest that dipping in a NaCl solution was effective in alleviating the browning issues of herbaceous peony tissue cultures, and had positive synergistic effects with PVP on browning suppression and callus induction.


Introduction
Herbaceous peony (Paeonia lactiflora Pall.) is a popular flower in Korea due to its attractive appearance, fragrance, large flowers, a wide range of colors, and high ornamental value. It is also one of the most famous and traditional flowers in China, where people have cultivated herbaceous peony

Treatments
Dipping in a NaCl (Yakuri Pure 103 Chemicals, Kyoto, Japan) solution, supplementing the medium with polyvinyl pyrrolidone (PVP) (Yakuri Pure 103 Chemicals, Kyoto, Japan), low temperature storage, and transfer of the explants to a new medium were the four methods used in this study. The details on the treatment combinations are listed in Table 1, and denoted as follows: Na means dipping petals in a 0.5 g·L −1 NaCl solution before planting; P means addition of 0.5 g·L −1 PVP to the medium; L means storage at 4 • C for 24 h after planting; NT means no transfer; T means transfer to a new medium after 24 h of planting. Treatments were set into 4 groups: a. group contains NT, T, and PNT; b. group contains NaNT, NaT, and NaPNT; c. group contains LNT, LT and LPNT; d. group contains NaLNT, NaLT, and NaLPNT. Dipping petals in a 0.5 g·L −1 NaCl solution before planting, then storage at 4 • C for 24 h after planting, and transfer to a new medium after 24 h of planting 12 NaLPNT Dipping petals in a 0.5 g·L −1 NaCl solution before planting, then storage at 4 • C for 24 h after planting, addition of 0.5 g·L −1 PVP to the culture medium, and no transfer to a new medium after 24 h of planting

Browning Evaluation
The browning of the cut edges of the petal explants was visually evaluated. The percentage of explants with browning was evaluated at 0 and 1 day, and 1, 2, 3, and 4 weeks after the initiation of culture. The extent of browning was denoted by S0 (no browning), S1 (slight browning), S2 (definite browning), and S3 (extreme browning).

Total Phenolic Contents
A total of 3 mg of tissue was homogenized in the mortar with 1.5 mL distilled water. The homogenate was centrifuged for 15 minutes at 12,000 xg. The supernatant was carefully transferred to a 1.5 mL centrifuge tube (10090, SPL, Pocheon, Republic of Korea). The Folin-Ciocalteu procedure was followed using a commercial reagent (Yakuri Pure 103 Chemicals, Kyoto, Japan) [34]. Then 0.4 mL of the supernatant was added into a tube containing 3.6 mL distilled water. After being shaken, a 0.4 mL Folin-Ciocalteu reagent was added. Following another shake, a 7% (w/v) Na 2 CO 3 solution was added after 5 minutes. After fully mixing, distilled water was added to the mixture to make the total volume 10 mL. After 90 minutes at room temperature avoiding light, absorbances at 765 nm were recorded by UV-spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). The phenol standards were prepared by dissolving 10 mg of gallic acid (Yakuri Pure 103 Chemicals, Kyoto, Japan) in water and diluting it to 50 mL. The Folin-Ciocalteu protocol was applied to the solution containing 0-0.06 mg·mL −1 gallic acid to construct the standard curve. Referred to Hisaminata's method [35], a 3 mg of tissue was homogenized in the mortar with a 1.5 mL MacIIvaine buffer (pH 6.5; a mixture of 0.1 M citric acid (Yakuri Pure 103 Chemicals, Kyoto, Japan) and 0.2 M disodium phosphate (Yakuri Pure 103 Chemicals, Kyoto, Japan)). The homogenate was centrifuged for 15 minutes at 12,000 xg. The supernatant was carefully transferred to a 1.5 mL centrifuge tube and used as a crude enzyme of PPO. The activity of PPO was measured spectrophotometrically at 410 nm to detect the decrease of chlorogenic acid (Yakuri Pure 103 Chemicals, Kyoto, Japan) as a substrate. The reaction solution consisted of 2 mL of MacIIvaine buffer (pH 6.0), 0.5 mL of 0.1 mol·L −1 catechol (Yakuri Pure 103 Chemicals, Kyoto, Japan), and 0.5 mL of the crude enzyme solution. A total of three 3-minute reads was made with a 15 second recording interval. An increase in the absorbance of 0.001 per minute at 30 • C was defined as one unit.

Phenylalanin Ammonia-Lyase (PAL)
Referred to Hisaminata's method [35], a 3 mg of tissue was homogenized in the mortar with a 1.5 mL 0.2 M boric acid-NaOH (Yakuri Pure 103 Chemicals, Kyoto, Japan) buffer (pH 8.8). The homogenate was centrifuged for 15 minutes at 12,000 xg. The supernatant was carefully transferred to a 1.5 mL centrifuge tube and used as a crude enzyme of PAL. The activity of PAL was measured spectrophotometrically at 290 nm to detect the increase in cinnamic acid as a product. The reaction solution consisted of 2.0 mL of distilled water, 1.0 mL of 0.02 mol·L −1 L-phenylalanine (Yakuri Pure 103 Chemicals, Kyoto, Japan), and 1.0 mL of the crude enzyme solution. The solution was placed in a 30 • C water bath for 1 h to react after being fully shaken. One unit of PAL activity was defined as the amount of enzyme that produced one micromole of cinnamic acid.

Statistical Analysis
Each treatment was replicated at least four times, and all data were subjected to an analysis of standard deviation (SD) using SAS (SAS Institute, Cary, NC, USA) and to the least significant difference (LSD) test. The significance was set at p ≤ 0.05 for comparing the means. Figure 1 shows the effect of treatments on the browning of petal explants of herbaceous peony during the initial stage of tissue culture. Figure 1a compares the effect of transferring the explants to a new medium and adding PVP to the medium on explant browning. The browning percentage increased rapidly to 90.7% at 1 day after culture initiation in the treatment in which the explants were not transferred to a new medium. Transferring to a new medium and adding PVP significantly reduced the browning percentage to 70.4 and 29.6%, respectively. At 1 day after culture initiation, browning appeared in most of the petal explants, and there were no significant differences in the browning percentage of the explants among the three treatments. Similar to those observed in this study, transferring to a fresh medium and adding PVP to the medium were reported to be practical methods to reduce the accumulation of browning compounds [19]. In this study, adding PVP was more effective than transferring to a new medium in reducing the browning, which may be due to the absorption of some browning compounds by PVP.

Effects of Treatments on Explant Browning
Plants 2020, 9, x FOR PEER REVIEW 6 of 13 methods to reduce the accumulation of browning compounds [19]. In this study, adding PVP was more effective than transferring to a new medium in reducing the browning, which may be due to the absorption of some browning compounds by PVP.  Figure 1b shows the effects on explant browning of dipping the explants in a 0.5 g·L -1 NaCl solution before planting, and a combination of the NaCl pretreatment, PVP addition and transferring to a new medium. At 1 day after culture initiation, there were no browned explants found in the NaNT and NaT, while the browning percentage of in the NaPNT reached to 77.8%. However, there were no significant differences in the browning of the explants among the treatments at 4 week after culture initiation. It was shown that browning could be effectively inhibited by dipping the explants in a NaCl solution. The NaCl has been commonly used to inhibit browning of apples. A strongly pHdependent PPO inhibitory effect was observed by NaCl [36]. However, when applied in conjunction with PVP, the effectiveness of NaCl in browning inhibition was reduced, which may be due to adsorption by PVP. Figure 1c shows the effect on explant browning of storing the explants at 4°C for 24 h after planting, and the combination of low temperature, PVP, and transferring to a new medium. At this low temperature, the browning percentages at 1 day after culture initiation of the explants transferred to a new medium, not transferred to a new medium, and PVP added were 64.8, 84.3, and 18.5%, respectively. Compared to the results shown Figure 1a, the low temperature storage was more effective in inhibiting browning. The low temperature storage has been used to inhibit browning in some vegetables. At 4 week after culture initiation, there were no significant differences in the browning of the explants among treatments. Figure 1d shows combined effects of NaCl pretreatment, PVP addition, low temperature storage, and transferring to a new medium. As the results in Figure 1b, regardless of whether the explants were transferred to a new medium or not, there were no browning explants observed at 1 day after culture initiation when the explants were dipped in a 0.5 g·L -1 NaCl solution before planting and  Figure 1b shows the effects on explant browning of dipping the explants in a 0.5 g·L −1 NaCl solution before planting, and a combination of the NaCl pretreatment, PVP addition and transferring to a new medium. At 1 day after culture initiation, there were no browned explants found in the NaNT and NaT, while the browning percentage of in the NaPNT reached to 77.8%. However, there were no significant differences in the browning of the explants among the treatments at 4 week after culture initiation. It was shown that browning could be effectively inhibited by dipping the explants in a NaCl solution. The NaCl has been commonly used to inhibit browning of apples. A strongly pH-dependent PPO inhibitory effect was observed by NaCl [36]. However, when applied in conjunction with PVP, the effectiveness of NaCl in browning inhibition was reduced, which may be due to adsorption by PVP. Figure 1c shows the effect on explant browning of storing the explants at 4 • C for 24 h after planting, and the combination of low temperature, PVP, and transferring to a new medium. At this low temperature, the browning percentages at 1 day after culture initiation of the explants transferred to a new medium, not transferred to a new medium, and PVP added were 64.8, 84.3, and 18.5%, respectively. Compared to the results shown Figure 1a, the low temperature storage was more effective in inhibiting browning. The low temperature storage has been used to inhibit browning in some vegetables. At 4 week after culture initiation, there were no significant differences in the browning of the explants among treatments. Figure 1d shows combined effects of NaCl pretreatment, PVP addition, low temperature storage, and transferring to a new medium. As the results in Figure 1b, regardless of whether the explants were transferred to a new medium or not, there were no browning explants observed at 1 day after culture initiation when the explants were dipped in a 0.5 g·L −1 NaCl solution before planting and being stored at 4 • C for 24 h. The browning percentage of the explants in the NaLPNT at 1 day after culture initiation was 55.6%, which was higher than that in the LPNT, but lower than that in the NaPNT. It seems that low temperature storage could reduce the negative effect of PVP to NaCl on browning inhibition. At 4 weeks after culture initiation, there were no significant differences in the browning percentage among treatments.
The effects of treatments on the browning of the explants observed at 4 weeks after culture initiation, graded on a scale of S0 (no browning), S1 (slight browning), S2 (definite browning), and S3 (extreme browning), are shown in Figure 2. For the explants not transferred to a new medium, the treatment of dipping in a 0.5 g·L −1 NaCl solution before planting, and the treatment of storing at 4 • C for 24 h after planting significantly delayed browning of the explants. The percentage of the explants at S1 was 40.3% and 44.4% for the above two treatments, NaNT and LNT, respectively. When these two treatments were applied in a combination, the browning inhibition was enhanced to have 66.7% of the explants at S1, 33.3% of them at S2, and none of them at S3 (Figure 2, NALNT). It seems that NaCl solution dipping and low temperature storage were the two most practical methods in suppressing tissue browning [36,37].
Plants 2020, 9, x FOR PEER REVIEW 7 of 13 being stored at 4 ℃ for 24 h. The browning percentage of the explants in the NaLPNT at 1 day after culture initiation was 55.6%, which was higher than that in the LPNT, but lower than that in the NaPNT. It seems that low temperature storage could reduce the negative effect of PVP to NaCl on browning inhibition. At 4 weeks after culture initiation, there were no significant differences in the browning percentage among treatments. The effects of treatments on the browning of the explants observed at 4 weeks after culture initiation, graded on a scale of S0 (no browning), S1 (slight browning), S2 (definite browning), and S3 (extreme browning), are shown in Figure 2. For the explants not transferred to a new medium, the treatment of dipping in a 0.5 g·L -1 NaCl solution before planting, and the treatment of storing at 4 ℃ for 24 h after planting significantly delayed browning of the explants. The percentage of the explants at S1 was 40.3% and 44.4% for the above two treatments, NaNT and LNT, respectively. When these two treatments were applied in a combination, the browning inhibition was enhanced to have 66.7% of the explants at S1, 33.3% of them at S2, and none of them at S3 (Figure 2, NALNT). It seems that NaCl solution dipping and low temperature storage were the two most practical methods in suppressing tissue browning [36,37]. When the explants were transferred to a new medium, 24.4%, 35.6%, and 40.0% of the explants were at S1, S2, and S3, respectively (Figure 2, T). Dipping the explants in a 0.5 g·L -1 NaCl solution before planting delayed the browning to a S3 level, while storing the explants at 4 ℃ for 24 h after planting accelerated the browning to a S3 level (Figure 2, NaT and LT). A combined treatment of dipping in a 0.5 g·L -1 NaCl solution before planting and storing at 4 ℃ for 24 h after planting resulted in the most of the explants being at S1 (31.5%) and S2 (46.3%) levels (Figure 2, NaLT).
When PVP was added to the medium, the browning was very effectively suppressed to have 63.9% of the explants at S1 (Figure 2, PNT), a much higher proportion than that of the NT or T. When these explants were dipped in a 0.5 g·L -1 NaCl solution before being planted, all explants were at S1 (Figure 2, NaPNT), representing a significant browning inhibition. However, storing the explants at When the explants were transferred to a new medium, 24.4%, 35.6%, and 40.0% of the explants were at S1, S2, and S3, respectively (Figure 2, T). Dipping the explants in a 0.5 g·L −1 NaCl solution before planting delayed the browning to a S3 level, while storing the explants at 4 • C for 24 h after planting accelerated the browning to a S3 level (Figure 2, NaT and LT). A combined treatment of dipping in a 0.5 g·L −1 NaCl solution before planting and storing at 4 • C for 24 h after planting resulted in the most of the explants being at S1 (31.5%) and S2 (46.3%) levels (Figure 2, NaLT).
When PVP was added to the medium, the browning was very effectively suppressed to have 63.9% of the explants at S1 (Figure 2, PNT), a much higher proportion than that of the NT or T. When these explants were dipped in a 0.5 g·L −1 NaCl solution before being planted, all explants were at S1 (Figure 2, NaPNT), representing a significant browning inhibition. However, storing the explants at 4 • C for 24 h after planting along with PVP addition to the medium increased the percentage of S2 and S3 explants to 37.5% and 25.6%, respectively (Figure 2, LPNT). A combination of dipping in a 0.5 g·L −1 NaCl solution before planting and storing at 4 • C for 24 h after planting resulted in 10.0, 72.0, and 18.1% of the explants being at S1, S2, and S3 levels, respectively (Figure 2, NaLPNT).
Compared to those explants not transferred to a new medium, transferring the explants to a new medium was no longer effective in inhibiting explants to be brown at 4 weeks after culture initiation, especially when in combination with NaCl, low temperature, or both. Adding PVP to the medium was a very effective method to control the progress of explant browning, especially when combined with the NaCl solution dipping. Low temperature storage weakened PVP's effectiveness of browning inhibition. Dipping in a NaCl solution before planting had a significant browning inhibitory effect, especially as combined with PVP addition. Furthermore, NaCl could adjust the native effect of low temperature storage. Hence, dipping in a NaCl solution before planting and PVP addition to the medium were the two most practical ways to inhibit browning and also to control the browning progress of the explants [21,36].

Effects of Treatments on Total Phenol Content and Enzyme Activities
As shown in Figure 3, the total phenol content of the explants in different treatments showed different trends. When the petal explants were stored at 4 • C for 24 h after planting, the total phenol content increased gradually to 1 day after culture initiation ( Figure 3c). This was consistent with the results in cauliflower heads [38] and fresh lettuce [35], but not with some outliers such as lotus root [39], which may be due to differences in the raw materials. Combining the NaCl dipping treatment with storage at 4 • C for 24 h after planting resulted in much lower total phenol contents of the explants than that of the explants without the NaCl solution dipping (Figure 3d). Different phenols are the main substrates for enzymatic browning in fruits and vegetables, and it seems that NaCl could inhibit the biosynthesis of total phenols [20]. Figure 3e-h shows the activities of enzyme PPO. It is obvious that transferring the explants to a new medium, dipping in a 0.5 g·L −1 NaCl solution before planting, storing at 4 • C for 24 h after planting, and adding PVP to the medium suppressed the PPO activity in the petal explants for the first two weeks. The PPO activity was the lowest in the explants when they were dipped in a 0.5 g·L −1 NaCl solution before planting, stored at 4 • C for 24 h after planting, and not transferred to a new medium. The PPO is a very important enzyme for the formation of the browning compound quinine [40,41]. Hence, transferring to a new medium, dipping in a 0.5 g·L −1 NaCl solution before planting, storing at 4 • C for 24 h after planting, and adding PVP to the medium are effective ways to delay explant browning by inhibiting the PPO activity.
The PAL is a key enzyme for the synthesis of phenolic compounds [42]. The PAL activities in the explants are presented in Figure 3i-l. The PAL activity increased to the peak at 0 day after culture initiation when the explants were dipped in a 0.5 g·L −1 NaCl solution before planting (Figure 3j,l). Without the NaCl solution dipping, the PAL activity peaked at 1 day after culture initiation ( Figure 3I,k). Low temperature storage reduced the PAL activity regardless of treatment with NaCl solution dipping or PVP addition to the medium (Figure 3k,l). This is consistent with the results of PAL activity in pineapple [43] and lotus [39]. However, the PAL was found not to be significantly correlated to the PPO and total phenol contents in this study. There may be other enzymes involved in the metabolism [44].  Figure 4a shows the contamination percentage of the explants observed at 8 weeks after culture initiation. The data clearly indicates that storage at 4 ℃ for 24 h after planting (LNT) had the greatest contamination percentage. Transferring the explants to a new medium may be a way to decrease the contamination percentage, because higher contamination percentages were recorded in the NT, NaNT, and NaLNT. As compared to the explants not transferred to new a medium, the contamination percentage of the explants in the T, NaT, LT, and NaLT was much lower. The PVP was reported as an effective browning inhibitor (Raghuvanshi and Srivastava 1995). In this study, adding PVP to the medium was also a good way to inhibit contamination, especially when it was combined with the NaCl solution dipping, since the lowest contamination percentage was observed in the NaPNT.

Effects of Treatments on Explant Contamination and Callus Induction
Callus induction is a key point of the success of many tissue cultures. The results of this study on callus induction are shown in Figure 4b. Dipping the explants in a 0.5 g·L -1 NaCl solution before planting significantly increased callus induction percentage. Adding PVP to the medium also enhanced callus induction. Combining PVP addition with dipping in a NaCl solution (NaPNT) gave the greatest callus induction percentage. However, when the explants were stored at 4 ℃ for 24 h after planting, the callus induction percentage decreased even along with NaCl solution dipping and PVP addition.  Figure 4a shows the contamination percentage of the explants observed at 8 weeks after culture initiation. The data clearly indicates that storage at 4 • C for 24 h after planting (LNT) had the greatest contamination percentage. Transferring the explants to a new medium may be a way to decrease the contamination percentage, because higher contamination percentages were recorded in the NT, NaNT, and NaLNT. As compared to the explants not transferred to new a medium, the contamination percentage of the explants in the T, NaT, LT, and NaLT was much lower. The PVP was reported as an effective browning inhibitor (Raghuvanshi and Srivastava 1995). In this study, adding PVP to the medium was also a good way to inhibit contamination, especially when it was combined with the NaCl solution dipping, since the lowest contamination percentage was observed in the NaPNT.

Effects of Treatments on Explant Contamination and Callus Induction
Callus induction is a key point of the success of many tissue cultures. The results of this study on callus induction are shown in Figure 4b. Dipping the explants in a 0.5 g·L −1 NaCl solution before planting significantly increased callus induction percentage. Adding PVP to the medium also enhanced callus induction. Combining PVP addition with dipping in a NaCl solution (NaPNT) gave the greatest callus induction percentage. However, when the explants were stored at 4 • C for 24 h after planting, the callus induction percentage decreased even along with NaCl solution dipping and PVP addition. Plants 2020, 9, x FOR PEER REVIEW 11 of 13

Conclusions
Browning is prevalent in tissue cultures of Paeonia lactiflora Pall. (herbaceous peony), and severely affects and restricts the growth and differentiation of the explants and/or success of tissue culture. In this study, dipping the excised explants in a sodium chloride (NaCl) solution as a pretreatment, adding polyvinyl pyrrolidone (PVP) to the medium, storing the planted explants at 4 ℃ for 24 h, and transferring planted explants to the same fresh medium after 24 h were all effective methods for suppressing browning of petal explants in tissue cultures of herbaceous peony 'Festival Maxima'. It was observed in all treatments that the decreased browning was accompanied with higher levels of phenols and lower activities of phenylalanine ammonia-lyase (PAL) and polyphenoloxidase (PPO). Overall, the results suggest that dipping the explants in a NaCl solution was not only effective in suppressing browning of the petal explants of herbaceous peony, but also had positive synergistic effects with PVP added to the medium on callus induction.