| Plant latin name | Paeonia lactiflora Pallas |
| Literature code | Paeonia_lactiflora-Ref-7 |
| Reference | Yu XN et al, African Journal of Biotechnology 11(41): 9776-9781 (2012) |
| Summary | Underground buds of herbaceous peony (Paeonia lactiflora Pall.) ‘Da Fu Gui’ were micropropagated in vitro. The basic processes including culture initiation, shoot induction, axillary shoot proliferation and rooting were established. The best initial medium of ‘Da Fu Gui’ was half-strength MS (Murashige and
Skoog) medium (double-strength Ca2+) supplemented with 0.5 mg l-1 6-benzylaminopurine (BA) plus 0.5
mg l-1 gibberellic acid (GA3). The best medium for axillary shoot induction was half-strength MS medium (double-strength Ca2+) supplemented with 1.0 mg l-1 BA plus 0.5 mg l-1 kinetin (Kin), while 0.5 mg l-1 BA +
0.3 mg l-1 Kin was best for shoot proliferation. Shoot height at the time of inoculation had a great effect on proliferation and growth of ‘Da Fu Gui’. Putrescine (Put) (0.5 to 5.0 mg l-1) prevented rooting of ‘Da Fu Gui’ but it favored the development of roots. Highest rooting percentage was observed on half-strength MS medium (double-strength Ca2+) supplemented with 1 mg l-1 indole-3-butyric acid (IBA). |
| Objectives | Micropropagation of herbaceous peony |
| Materials | An excellent (highly responsive to manipulation) herbaceous cultivar ‘Da Fu Gui’, which is a traditional Chinese variety with
high ornamental value and vigorous growth in vitro |
| Explant | Two-year-old underground buds were collected in spring from the
Society of Forestry Experimental Station. The buds were about 3
cm in length and were collected between the closed and sprouting
stage. |
| Initial culture | Before inoculating, underground buds were washed in tap water for 30 min. Outer scales were peeled off and denuded buds were soaked in 75% ethanol for 30 s followed by 10 min sterilization with a dilute solution of HgCl2 (0.1% w/v). Buds were rinsed five times (5 min each time) in autoclaved distilled water, then cultured in 100-ml Erlenmeyer flasks containing 30 ml of agar-solidified medium with one bud per flask. All explants were placed on agar (0.7%) solidified medium. The basal medium for all experiments was 1/2 MS with double-strength calcium chloride (Ca2+) supplemented with 30 g L-1 sucrose. The effect of GA3 (0.5 mg l-1) combined with BA (0.5, 1.0, 1.5 and 2.0 mg l-1) on initiation culture of ‘Da Fu Gui’ was tested. The pH of all media was adjusted to 5.8 prior to autoclaving at 118°C for 18 min. Culture vessels were placed at 25 ± 2°C in a 14-h
photoperiod with 50 μmol m-2s-1 PPFD using cool white fluorescent tubes. Data were collected after 30 days of culture. The explants were subcultured every 30 days. The initiation medium with 0.5 to 1.5 mg l-1 BA favored the growth of underground buds the most, with a 100% sprouting percentage in all three treatments. The highest leaf expansion percentage (96.97%) and height (3.61 cm) was observed with 1.0 mg l-1 BA + 0.5 mg l-1 GA3. The slightly higher number of lateral shoots (2.42) and the best growth of shoots were noted on medium containing
the lowest concentration of BA (0.5 mg l-1). A
higher concentration of BA (2.0 mg l-1) did not further stimulate the growth of shoots, resulting in the lowest sprouting percentage (78.90%), leaf expansion percentage (66.27%) and number of lateral shoots (1.72). In
addition, a higher concentration of BA inhibited explant growth, induced hyperhydricity and deformed shoots. Thus, medium containing 1.0 mg l-1 BA + 0.5 mg l-1 GA3 was the best choice for initial shoot induction of ‘Da Fu Gui’, considering the higher number (2.28) of lateral shoots. |
| Shoot multiplication | The effect of different treatments on axillary shoot induction was tested. Shoot induction medium was 1/2 MS + 1.0 mg l-1 BA, 1/2 MS
+ 1.0 mg l-1 BA + 0.5 mg l-1 Kin, 1/2 MS + 1.0 mg l-1 BA + 0.5 mg l-1 Kin + 0.1 mg l-1 IBA, 1/2 MS + 1.0 mg l-1 BA + 0.5 mg l-1 Kin + 0.1 mg l-1 IAA. There were two experiments for axillary shoot proliferation. Firstly, the effects of different treatments on shoot proliferation were tested. The treatments were 1/2 MS + 0.5 mg l-1 BA + 0.3 mg l-1 Kin, 1/2 MS + 0.5 mg l-1 BA + 0.3 mg l-1 Kin + 500 mg l-1 casein hydrolysate (CH), 1/2 MS + 0.5 mg l-1 BA + 0.3 mg l-1 Kin + 0.2 mg l-1 IBA. Then, the effects of initial height of shoots used in inoculation on the proliferation and growth of ‘Da Fu Gui’ were tested. The shoots were divided into three groups according to height (1, ≤1 cm; 2 = 1 to 2 cm; 3, ≥ 2 cm) before culture. The medium was 1/2 MS supplemented with 0.2 mg l-1 BA or plant growth regulator (PGR)-free 1/2 MS medium. Axillary shoots were able to generate after three weeks of culture. After 20 days of culture, a large number of axillary shoots (about 10) were produced from meristematic regions and from the base of buds. Medium with 1.0 mg l-1 BA + 0.5 mg l-1 Kin resulted in the highest shoot induction percentage (58.93%). Most axillary shoots (12.29) and the lowest shoot induction percentage (37.30%) were observed with 1.0 mg l-1 BA. The addition 0.1 mg l-1 IBA or 0.1 mg l-1 IAA to medium containing 1.0 mg l-1 BA and 0.5 mg l-1 Kin decreased the number of axillary shoots. In conclusion, medium containing 1.0 mg l-1 BA + 0.5 mg l-1 Kin favored axillary shoot induction the most. In the case of axillary shoot proliferation, a slightly higher number of multiple shoots (3.3) was obtained on medium with 0.5 mg l-1 BA + 0.3 mg l-1 Kin. The addition of 500 mg l-1 CH or 0.2 mg l-1 IBA did not favor shoot proliferation. Thus, the best
proliferation medium for ‘Da Fu Gui’ was 0.5 mg l-1 BA + 0.3 mg l-1 Kin. In the case of the shoot height effect, most leaves (7.95), greatest height (2.96 cm) and percentage of sturdy shoots (79.90%) were obtained in shoots taller than 2 cm cultured on PGR-free 1/2 MS medium. Shoots taller than 2 cm
significantly resulted in the best effect followed by shoots 1 to 2 cm in size, then shoots smaller than 1 cm, with or without BA. The addition of BA to medium resulted in poor growth of shoots, gradual hyperhydricity and etiolation. Shoots easily became dormant and fragile in summer because of high humidity, so, even a low concentration of BA (0.2 mg l-1) harmed shoot subculture. Thus, during the hot summer months, shoots taller than 2 cm cultured on PGR-free 1/2 MS medium were optimal for subculture. |
| Rooting | In the fifth experiment, the effects of Put (0, 0.5, 1.0, 2.0, 3.0 and 5.0 mg l-1) combined with 1 mg l-1 IBA on root induction of ‘Da Fu
Gui’ were tested. At the rooting stage, shoots were placed on different rooting media for 20 days and then transferred to PGR-free
1/2 MS medium containing 0.2% activated charcoal (AC). The shoots used for the proliferation and rooting were 2 cm long
except shoots for the rooting induction experiment when the effect
of Put was 2.0 mg l-1. Roots were produced at the base of shoot stems after shoots were transferred to PGR-free 1/2 MS medium
containing 0.2% AC for 20 days. The highest rooting percentage (28.15%) was observed on medium with 1.0 mg l-1 IBA + 0 mg l-1 Put. Even though the percentage rooting decreased when Put was added, the quality of roots and shoots was good, that is, sturdy and without hyperhydricity. The highest number of roots (4.50), root length (2.07 cm), number of leaves (5.05) and height (2.80 cm) were observed on medium with 1.0 mg l-1 IBA + 2.0 mg l-1 Put. Higher concentrations of Put (3.0 to 5.0 mg l-1) negatively affected root induction. Put thus does not induce roots but enhances rooting. |
| Acclimation | |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | |
| Ingredients analyzed | |
| Extraction | |
| Analitical methods | |
| Notes | |