| Plant latin name | Paeonia lactiflora Pallas |
| Literature code | Paeonia_lactiflora-Ref-4 |
| Reference | Jana S et al., Science International 1: 318-324 (2013) |
| Summary | Background: Peony is an ornamental as well as medicinal plant which is difficult to propagate in vitro. Its major problem during in vitro culture is leaching out of phenols in the medium. It causes hindrance in growth and
development. Rooting is a complex process and main step for vegetative propagation. The main objective is to propagate this plant and produce multiple copies and understand rooting phenomenon which is usually complicated. Methods: The underground rhizomatous buds of Paeonia lactiflora Pall. ‘Hortensis’ were selected as the explants.
In vitro micropropagation was carried out along with biochemical studies during rooting process. Results: Shoot induction, axillary shoot proliferation and rooting were established. The best initial medium for ‘hortensis’ was the half-strength Murashige and Skoog (MS) medium (double-strength Ca+2) supplemented with 1 mg L-1 N6 benzyl-adenine (BA) and 0.5 mg L-1 Gibberellic Acid (GA3). Shoot induction was 100% with 6.2 shoots per explant. Rooting was also observed on the half-strength MS medium supplemented with 1.0 mg L-1 Indole-3-butyric Acid (IBA) and indole-3-acetic acid (IAA). The highest percentage of rooting (15%) was observed when in vitro shoots were dipped in the full-strength liquid MS medium supplemented with 10.0 mg L-1 IBA without agar for five days and then transferred to the quarter strength MS medium without any Plant Growth Regulator (PGR). Conclusion: The cultivar P. lactiflora hortensis is hard to root in vitro. The activities of peroxidase, indole acetic oxidase, polyphenolic oxidase and phenolic content have been estimated prior to and after rooting. These enzyme activities and phenolics have been reported to play a major role in rooting. |
| Objectives | In vitro propagation of herbaceous peony to meet the constantly growing demand of the global flower market |
| Materials | Paeonia lactiflora ‘hortensis’ |
| Explant | Underground rhizotomous buds of Paeonia lactiflora ‘hortensis’ were collected in spring from the field. The buds were 3-5 cm in length and were in between the closed and sprouting stage. |
| Initial culture | The collected buds were washed under running tap water with few drops of Tween 20 for 10 min. The outer scales were peeled off and buds were soaked in 70% (v/v) ethanol (Yakuri Pure Chemicals, Kyoto, Japan) for 5 min and then rinsed with distilled water. After this, they were soaked in 1.5% (v/v) NaOCl (Yakuri Pure Chemicals, Kyoto, Japan) for 5 min and the rinsed with distilled water. Under aseptic conditions, 3-5 scales were removed and treated with 70% (v/v) ethanol and 1.5% (v/v) NaOCl for 5 min each and then rinsed with sterile distilled water thrice. All sterilized buds were cultured on the medium (1/2MS salts, vitamins, double-strength CaCl2, 3% sucrose and plant growth regulators (PGRs), solidified with 0.8% agar, 0.1% w/v activated charcoal, 50 mL medium/300 mL Magenta box, 1 bud/box, 25±2℃, 16 h photoperiod: 30 µmol m-2sec-1 PPFD). The effect of giberellic acid (GA3), N6-benzyl-adenine (BA), Kinetin (Kin), Indole-3-butyric Acid (IBA) and Indole-3-acetic Acid (IAA) was tried on initiation culture of ‘hortensis’. Their effect was observed on shoot induction and proliferation of cultures. The surface sterilization method helped to produce 85% aseptic cultures. After inoculation of underground rhizomatous buds on the medium, they grew quickly within 15 days with 4-5 leaves. These buds sprouted out into main shoots and lateral shoots. Thirty days later, one to several new shoots developed from each bud eye region. The highest shoot induction rate (100%) was achieved with 1.0 mg L-1 BA and 0.5 mg L-1 GA3 . The highest multiplication rate with 6.2 shoots per explant was also obtained on the medium with the same concentrations of BA and GA3. |
| Shoot multiplication | Elongated shoots obtained from the in vitro cultures were cut from the nodal parts and transferred to fresh medium containing the half-strength MS medium supplemented with 1.0 mg L-1 BA and 0.3 or 0.5 mg L-1 GA3 for axillary shoot multiplication. Application of GA3 showed a significant effect on shoot induction and elongation on cultures of P. lactiflora. The combination of BA and GA3 is used for formation and growth of axillary buds. |
| Rooting | The effect of IBA or IAA alone was tested on rooting of ‘hortensis’. The shoots were transferred to the half-strength MS medium with double-strength calcium chloride supplemented with either IBA or IAA at 0.5 or 1.0 mg L-1. The half-strength MS medium supplemented with IBA and IAA showed good number of roots. Reducing the MS salt concentration to a half level was found to be effective in terms of frequency of root induction. However, shoots became slightly withered and leaves turned yellow, probably due to long (30 days) exposure to activated charcoal. New rooting treatments have been developed, examining the effects of plant growth regulators by a short exposure to a solution with a high auxin concentration. Shoots were dipped in the full-strength liquid MS medium supplemented with 10.0 mg L-1 IBA without agar for 5 days and then were transferred to a quarter strength MS medium without any PGR. Thus, the highest rooting percentage (15%) was observed in the treatment, containing 0.1% (w/v)
activated charcoal. The highest
length of roots obtained was 3.6 cm. However, the plant turned yellow after few days. |
| Acclimation | The shoots with optimal height and well-developed
root length of 3.0 cm were hardened in a commercial
medium (Tosilee medium, Shinan Grow, Jinju, Korea). Unfortunately, none of the plants could survive. Within five days they all wilted and died. |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | |
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