| Plant latin name | Paeonia lactiflora Pallas |
| Literature code | Paeonia_lactiflora-Ref-5 |
| Reference | Albers MRJ et al, Acta Horticulturae 314: 85-92 (1992) |
| Summary | We developed a micropropagation protocol for Paeonia. Surface sterilized explants with one node were placed on a medium based on Lepoivre. The contamination percentage varied between 22-100 for the herbaceous peonies, but was negligible for the tree peonies. The multiplication rate was 1.3 to 2.9 in ca 7 weeks, at 15 ℃, a light intensity of 35 µmol/s/m2 and a daylength of 16 hrs. We examined several factors in order to improve the multiplication rate. None of these factors appeared to increase the multiplication
rate significantly. The only significant effect on the length of the shoots was a cold treatment. Rooting was better with IAA or IBA than with NAA, the optimal concentration being about 0.1 mg/l. Rooting in the
dark was better than in white or red light. About 50% of the plantlets was lost due to infection during weaning. Non-infected plantlets grew well. The first plants have just been planted outside, after one growing season in the greenhouse. |
| Objectives | Establishment of a quick micropropagation system for the multiplication of virus-free stock material or new cultivars in peonies |
| Materials | Paeonia lactiflora 'Sarah Bernhardt', P. lactiflora 'Karl Rosenfleld', P. officinalis
'Rubra Plena' and two unnamed cultivars of P. suffruticosa |
| Explant | Underground rhizotomous buds of Paeonia lactiflora |
| Initial culture | Rhizomes were immerged in a solution of
fungicide (2-5 g/l benomyl, 0.5 hr) and subsequently cultured at 20°C in the dark in plastic trays in 1-2 cm water with or without perlite. Twice a week they were sprayed with fungicide (0.5 g/l iprodion or 0.5 g/l
eupareen M) and a mixture of antibiotics (3 x 50 irg/l rifampicine, trimethoprim and chlorotetracycline). Sprouted buds were cut into nodal sections of 1.5-3 cm, and rinsed successively in running tap water (5-10 min) , a soap solution (20-40 ml/l Burtan; for 1 min), 96% ethanol (1 min.), tap water (1 min.) and 5%
CaOCl with a few drops of Tween 20 (10 min). Thereafter, the buds were rinsed three times in sterilised demineralized water, followed by a rinse in a sterile solution of ascorbic acid (10 g/l) + citric acid (10 g/l)
to supress oxidation at the cutting surface, and then once more in water. The nodal sections were dissected into segments with one bud and placed on a solidified multiplication medium (15 ml in culture tubes of 24 mm x 150 mm). The antibiotics (20 mg/l rifampicine and 20 mg/l trimetroprim) were added to the iniciation medium. In Paeonia lactiflora and Paeonia officinalis the percentage of contamination was 22-100, but in the two Paeonia suffruticosa cultivars it was negligible. |
| Shoot multiplication | The standard multiplication medium consisted of full strength Lepoivre macro- and microelements, 40 mg/l FeNaEDTA, 1.0 mg/l thiamine-HCl, 100 mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine-HCl, 1 mg/l BAP, 0.1 mg/l GA3 and 20 g/l saccharose. The pH was adjusted to 5.5 with 1.0 N KOH, prior to adding the agar (6 g/l) and autoclaving (15 min. at 120ºC). The explants were cultured at 15ºC, a light intensity of 35 µmol/s/m2 and a day length of 16 hrs. The explants were subcultured every 6-9 weeks. All long leaves were cut of for transplantation, and the explants were divided in clusters of 1-4 buds depending of their size. Most of the experiments were carried out with P. lacdflora 'Sarah Bernhardt'. To improve the multiplication rate, we examined the effect of: temperature, kinetine, BAP, 2IP, GA3, activated charcoal, liquid medium, day length, sugar, cold treatment, addition of a small concentration of auxin and concentration of macroelements. At least 30 cubes were used for each treatment. At the end of a subculture cycle we determined both the increase of the number of buds and the multiplication rate, i.e. the number of tubes in a subculture cycle divided by the number of tubes in the previous subculture cycle. Cold treatments (4ºC) before culture had no effect on the number of buds, the shoot length increased 10 fold. The multiplication rate usually varied between 1.3 to 2.9. Shortly after initiation, cultivation at 20ºC was
compared to that at 15ºC, in light as well as in the dark. At 20ºC the multiplication rate decreased in 4 subcycles from 2.7 to 0.6. At 15ºC the lowest multiplication rate was 1.33. Therefore all subcultures were done at 15ºC. None of the other factors examined appeared to increase the multiplication rate significantly.
In liquid media, the explants elongated but also became vitrified. |
| Rooting | The rooting medium was similar to the multiplication medium with the exception for the hormones. Instead of BAP and GA3, auxin was added instead. To be quite certain that the explants were not dormant, all explants received a cold treatment for at least 4 weeks at 4ºC in the dark. Explants of a similar size (2 to 4 buds) were placed on media containing IAA, IBA or NAA in concentrations of 0.1, 0.2, 0.5, 1.0, or 2.0 mg/l. All the explants were kept in the dark at 15ºC for the first two weeks, then they were placed in the light. The rooting percentage and the number of roots per rooted shoot were recorded every 2 weeks up to 12 weeks. In a next experiment a wider range of IBA was tested. Other factors examined were: different periods of cold, cultivation temperature, sugar concentration and colour of light. The two lowest concentrations of auxin produced the best results. The first roots appeared 6 weeks after transfer to the rooting media, and up to 10-12 weeks new roots were still being
formed. Rooting with IAA or IBA was better then with NAA. At a concentration higher than 0.5 mg/l the explants formed abundantly callus, and the quality of the explants was reduced. However, a low concentration of auxin was required for rooting. The optimal concentration for IBA was approximately 0.1 mg/l. The number and quality of the roots at this concentration was better than at higher
concentrations. The effect of varying periods of cold and different cultivation temperatures, was not clear. Saccharose 30 g/l produced the highest rooting percentage. Rooting in the dark was better than rooting in white or red
light. Off all the experiments the best result was obtained after 12 weeks on standard medium (0.1 mg/l IBA) at 12.5ºC in the dark (80% rooting). |
| Acclimation | Rooted plantlets from several rooting experiments were used in acclimatization experiments. After cutting
back the shoots of the plantlets to a length of 2-3 cm, they were immerged in a solution of 20 g/l benomyl for 2-4 min. Thereafter they were planted in a mixture of fertilized peat/sand (1:1) or peat/perlite (1:1) in
plastic trays with a transparant plastic cover. Cultivation occurred at 5, 10 or 15ºC , at a light intensity 45
µmol/s/m2 (16 hrs per day). The plants were sprayed with fungicide twice a week, any contaminated explants were discarded.
In a second experiment the day length (8, 12 or 16 hrs) was varied instead of temperature. To decrease the percentage of infection the plunge in the solution of benomyl was extended to at least 5 min. After planting in soil, more than 50% of the plants was lost due to infection. Growth in peat/ perlite during
the first 3-4 weeks was better than in peat/sand. No significant effect of temperature was found. Increased day length during the first 3 weeks resulted in increased shoot length and increased leaf size. With
extended weaning, the differences in shoot length disappeared whereas the differences in leaf size
remained. The non-infected peonies grew well and the root system developed quickly. |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | |
| Ingredients analyzed | |
| Extraction | |
| Analitical methods | |
| Notes | |