| Plant latin name | Paeonia lactiflora Pallas |
| Literature code | Paeonia_lactiflora-Ref-6 |
| Reference | Yu XN et al, International Journal of Plant Developmental Biology 6: 51-56 (2012) |
| Summary | Underground buds of herbaceous peony (Paeonia lactiflora Pall.) ‘Zhong Sheng Fen’ were used as explants for axillary shoot induction while stems, petioles and leaves of ‘Da Fu Gui’ and ‘Tao Hua Fei Xue’ were used as explants for callus induction. The effects of different basal media and concentrations of plant growth regulators (PGRs) on induction were investigated to establish an aseptic regeneration system. The best medium to induce and proliferate shoots was modified half-strength Murashige and Skoog (MS) medium with double
the concentration of Ca2+ supplemented with 1 mg l-1 gibberellic acid (GA3) plus 1 mg l-1 6-benzyladenine (BA). Two successive steps
were adopted for rooting shoots. Shoots were first cultured on Woody Plant Medium (WPM) plus 0.5 mg l-1 1-naphthyleneacetic acid
(NAA) for 10-15 days in the dark, then shoots were transferred to PGR-free WPM medium containing 0.2% activated charcoal; in this
case, rooting could reach 50%. The best explants for callus induction were young stems, and the best basal medium for callus induction was WPM for ‘Da Fu Gui’ and ‘Tao Hua Fei Xue’. |
| Objectives | Micropropagation of herbaceous peony |
| Materials | Three herbaceous cultivars
(which have high ornamental value and vigorous growth in
vitro) as experimental materials, ‘Zhong Sheng Fen’, ‘Da
Fu Gui’ and ‘Tao Hua Fei Xue’. |
| Explant | Four types of explants were investigated in this study: underground buds of ‘Zhong Sheng Fen’, and stems, petioles and leaves of ‘Da Fu Gui’ and ‘Tao Hua Fei Xue’. Underground buds of ‘Zhong Sheng Fen’ were thicker than other cultivars, so they were better for shoot induction, and were thus selected for our study. Buds were collected in winter while other explants were collected
in spring. All material was obtained from the Jiu Feng Herbaceous
Peony Experimental Base of Beijing Forestry University. |
| Initial culture | Before inoculating, buds were washed in
tap water for 30 min. Outer scales were peeled off and buds were soaked in 75% ethanol for 30 s followed by 15 min of sterilization with a diluted solution of sodium hypochlorite (2% active chlorine). Plant material was rinsed three times for 5 min in autoclaved distilled water (ADW). The remaining scales were peeled off, and
buds were soaked in a diluted solution of sodium hypochlorite (1% active chlorine) for 12 min. The material was rinsed in ADW four
times then cultured in 100-ml Erlenmeyer flasks containing 30 ml agar solidified medium with one or two buds per flask. Other explants (i.e., stems, petioles and leaves) were washed in tap water for 30 min
and cut into 2-4 cm long sections. Sections were soaked in 75% ethanol for 30 s followed by 15 min sterilization with a diluted
solution of sodium hypochlorite (0.5% active chlorine). Plant material was rinsed three times, 5 min each, in ADW. After sterilization,
explants were cut into shorter (1-2 mm) sections and cultured in Petri dishes (90 mm diameter) containing 30 ml of solidified
agar (0.7%) medium. 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-2 s-1 PPFD using cool white fluorescent tubes. For callus induction, cultures were placed in complete
darkness. Data were collected after 30 days of culture. To initiate culture, shoot tips of underground buds of ‘Zhong
Sheng Fen’ were cultured in half-strength MS medium (doublestrength Ca2+) supplemented with 1 mg l-1 GA3 + 1 mg l-1 BA or 1 mg l-1 GA3 + 1 mg l-1 BA + 0.1 mg l-1 NAA. For callus induction, stems and petioles were cut into 1-2 mm segments. The edges of leaf laminae were trimmed and the
remaining leaf tissue was divided into 5 mm2 squares and placed abaxial side down on the surface of media. Explants were cultured
on half-strength MS (double-strength Ca2+) or WPM medium supplemented with different PGRs as follows: (1) half-strength MS (double-strength Ca2+) + 2 mg l-1 BA + 0.2 mg l-1 NAA + 0.2 mg l-1 2,4-D; (2) half -strength MS (double-strength Ca2+) + 2
mg l-1 BA + 0.2 mg l-1 NAA; (3) WPM + 2 mg l-1 BA + 0.2 mg l-1 NAA + 0.2 mg l-1 2,4-D; (4) WPM + 2 mg l-1 BA + 0.2 mg l -1 NAA. The growth regulators, concentrations and combinations were selected on the basis of the domestic and foreign researches in tissue culture of peony. After inoculation, yellowish-white buds turned green in the light. The shoots elongated and leaves expanded with the axillary bud sprouting. GA3 (1 mg l-1) + BA (1 mg l-1) stimulated the growth of shoots whose stems were sturdy and leaves were large. |
| Shoot multiplication | In shoot proliferation culture, vigorous tissue-cultured plants were separated at the base
into 2-3 plantlets, and then placed on half-strength MS (doublestrength Ca2+) supplemented with 1 mg l-1 GA3 + 1 mg l-1 BA, 1 mg l-1 GA3 + 0.1 mg
l-1 Thidiazuron (TDZ), 1 mg l-1 BA or 0.2 mg l-1 BA. The highest number of axillary shoots (3.9) was obtained on medium with 1 mg l-1 GA3 + 0.1 mg l-1 TDZ, however, TDZ had a negative effect on the growth of shoots, which gradually became abnormal. Leaves were twisted and the plants tended to become hyperhydric. Most of the axillary shoots induced by TDZ could not be used in subsequent culture and proliferation percentage was lowest. TDZ was better than BA for shoot proliferation. The highest proliferation rate (2.3) and good quality
(i.e., vigorous growth, strong stems, dark-green leaves) shoots were obtained on medium with 1 mg l-1 GA3 + 1 mg
l-1 BA; 1 mg l-1 of BA was more effective than 0.2 mg l-1 in terms of shoot proliferation. GA3 had no significant effect on shoot multiplication. |
| Rooting | At the rooting stage, shoots were placed on different rooting medium for 10-15 days, and then transferred to PGR-free WPM medium containing 0.2% activated charcoal (AC). The rooting media were WPM (PGR-free), WPM + 0.5 mg l-1 Indole-3-butyric acid (IBA), WPM + 1 mg l-1 NAA, or WPM + 0.5 mg l-1 NAA. The highest rooting percentage (50%) was obtained on medium with 0.5 mg l-1 NAA in which the average root number was 2.3. Even though NAA induced
callusing at the base of shoots, roots were not connected to the callus, only to the stems, i.e. adventitious roots. Other media induced lower rooting percentages and fewer
roots. Roots could also be induced in auxin-free medium but, among all media, parameters were lowest in this case. |
| Acclimation | |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | |
| Ingredients analyzed | |
| Extraction | |
| Analitical methods | |
| Notes | |