M.R.J. Albers, B.P.A.M. Kunneman


Acta Horticulturae 1992, 314, pp. 85-92

M.R.J. Albers, Central Research Laboratory for Tissue culture of Horticultural Crops/Research Station for Nursery Stock PO Box 85 2160 AB Lisse, The Netherlands         
B.P.A.M. Kunneman Research Station for Nursery Stock PO Box 118,2770 AC Boskoop, The Netherlands



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 °C, a light intensity of 35 mmol/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.

I. Introduction

Peonies are hardy herbaceous or shrubby perennials of economic importance (van Dijk, 1988). In the Netherlands, the expansion of exports has  created a growing interest in Paeonia. Conventional propagation by division of the rhizome into parts with three to five eyes, however is very slow. A quick micropropagatlon system would overcome this problem and could also be used for the multiplication of virus-free stock material or new cultivars. Literature on the micropropagation of peony is limited. Radtke (1983) concluded that the main obstacle to successful tissue culture are exuded phenolic compounds, which inhibit growth. Hosoki et al. (1989) described in vitro propagation of herbaceous peony by a longitudinal shoot-split method. Harris and Mantell (1991) investigated the effect of subculture durations on the multiplication and rooting capacity of tree peonies. We report here on the successive phases of the micropropagation of Paeonia lactiflora 'Sarah Bernhardt', P. lactiflora 'Karl Rosenfleld', P. officinalis 'Rubra Plena' and two unnamed cultivars of P. suffruticosa (tree peony). Some of the results have been published before (Kunneman and Albers, 1989)

2. Material and Methods

2.1 Initiation

Contamination rates usually are rather high, because the buds of the herbaceous peonies grow sub-soil. Therefore an extended decontamination method was applied. 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.

2.2 Multiplication

The standard multiplication medium consisted of full strength Lepoivre (Quoirin et al., 1977) 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 BBL granulated) and autoclaving (15 min. at 120°C). The explants were cultured at 15°C, a light intensity of 35 mmol/s/m² (Philips TLD 33) 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 (fig 2). 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 auxine 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.

2.3. Rooting

The rooting medium was similar to the multiplication medium with the exception for the hormones. Instead of BAP and GA., 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 4°C 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 15 °C for the first two weeks, then they were placed in the light (conditions see multiplication). 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.

2.4 Acclimatization

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 15°C , at a light intensity 45 mmol/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 10 weeks the plants were transplanted to 0.75 l containers in the greenhouse and 6 months later to 1.5 l containers. After one growing season they were planted outside.

3. Results

3.1 Initiation

In Paeonia lactiflora and Paeonia officinalis the percentage of contamination was 22-100, but in the two Paeonia suffruticosa cultivars it was negligible.

3.2 Multiplication

Figure 1 shows that although cold treatments (4 C) 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 20 °C was compared to that at 15 °C, in light as well as in the dark. At 20 °C the multiplication rate decreased in 4 subcycles from 2.7 to 0.6. At 15 °C the lowest multiplication rate was 1.33. Therefore all subcultures were done at 15 C. None of the other factors examined appeared to increase the multiplication rate significantly. In liquid media, the  explants elongated but also became vitrified.

3.3 Rooting The two lowest concentrations of auxin produced the best results (fig.3). 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 rng/l the explants formed abundantly callus, and the quality of the explants was reduced. However, a low concentration of auxin was required for rooting (fig. 4). 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 12.5 C in the dark (80% rooting)

3.4 Acclimatization

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.

4. Discussion

To prevent contamination during initiation of cultures of Paeonia certain precautions need to be taken. The two most important factors in achieving aseptic cultures are the year of initiation and the cultivar. Tree peonies are generally less contaminated, due to the fact that their axillairy buds are not sub-soil. Explants grow slowly at the start. We were unable to improve the multiplication rate by changing common tissue culture factors. Only temperature had a clear effect. Preliminary experiments suggest that a cold treatment at the end of a subcycle, may increase multiplication. This indicates that one of the factors causing slow propagation is dormancy. In agreement with this, fluridone, an inhibitor of ABA-synthesis, also increases multiplication (Albers unpublished observation). Although some of the explants excrete phenolic compounds this did not seem to inhibit their growth, compared to explants without excretion. Thus, the statement of Radtke that exuded phenolic compounds inhibit growth, seems to be unjustified.

The regeneration of roots requires a long period of time on rooting medium. After some rooting experiments, the non-rooted explants were stored at 4°C and formed roots during this period. The acclimatization of Paeonia is sensitive to fungi and bacteria, which spread quickly among the plantlets. Individual acclimatization in separate pots or multipots might reduce fungal development.

Figure 1 - The effect of a cold pre-treatment at 4°C for 0, 2,5, 5 or 10 weeks on the increase of bud number and shoot length of Paeonia lacciflora 'Sarah Bernhardt'.



Figure 2 - Multiplication occurred by dividing the explant in sections with 1-4 buds. The shoots were cut back to a length of 1-2 cm. The top sections of some of the explants could also be uised (two upper explants).



Figure 3 - The effect of auxin on rooting of Paeonia lactiflora 'Sarah Bernhardt'.



Figure 4 - The effect of IBA concentration on rooting of Paeonia lactiflora 'Sarah Bernhardt'.



Figure 5 – Two rooted explants of Paeonia lactiflora 'Sarah Bernhardt'.



Figure 6 – Plant of Paeonia lactiflora 'Sarah Bernhardt', one year after acclimatization.


The authors would like to thank P. v.d.Voor A. de Regt and H. van Hoof for their technical assistence and dr. G.. de Klerk for reading the manuscript.


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