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

Abstract

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.

Keywords: Tissue culture; herbaceous peony; phenolic; phenylalanin ammonia-lyase (PAL); polyphenol oxidase (PPO).

Figure 1

Browning percentage of the petal explants of herbaceous peony ‘Festival Maxima’. The different abbreviations denote as follows: Na, dipping in a 0.5 g·L⁻¹ NaCl solution before planting; P, 0.5 g·L⁻¹ polyvinyl pyrrolidone (PVP) addition to the medium; L, storage at 4 °C for 24 h after planting; NT, not transferring to a new medium; and T, transferring to a new medium at 24 h after planting. Treatments were set into 4 groups; (a), NT, T, and PNT; (b), NaNT, NaT, and NaPNT; (c), LNT, LT, and LPNT; and (d), NaLNT, NaLT, and NaLPNT. Different alphabet letters indicate differences between treatments (ANOVA and Duncan’s test, p ≤ 0.05).

Figure 2

The extent of browning of the petal explants observed at 28 days after culture initiation: S0, no browning; S1, slight browning; S2, definite browning; and S3, extreme browning. Refer to Figure 1 for details on the different denotations.

Figure 3

Total phenol contents (a–d), and activities of polyphenol oxidase (PPO) (e–h) and phenylalanine ammonia-lyase (PAL) (i–l) of peony petal explants. Refer to Figure 1 for details on the different denotations. Different alphabet letters indicate significant differences between treatments (ANOVA and Duncan’s test, p ≤ 0.05).

Figure 3

Total phenol contents (a–d), and activities of polyphenol oxidase (PPO) (e–h) and phenylalanine ammonia-lyase (PAL) (i–l) of peony petal explants. Refer to Figure 1 for details on the different denotations. Different alphabet letters indicate significant differences between treatments (ANOVA and Duncan’s test, p ≤ 0.05).

Figure 4

The percentage of explant contamination (a) and callus induction (b) of peony petal explants observed at 56 days after culture initiation. Refer to Figure 1 for details on the different denotations. Different alphabet letters indicate significant differences between treatments (ANOVA and Duncan’s test, p ≤ 0.05).