Establishment of in vitro cultures of tree peonies

Decontamination of elite dwarf cultivars of the tree peony was increased by immersion of explants (leaves, buds, roots, seeds) in a mixture of 20% ethanol and 0.075% HgCh for 7min. Seeds were best decoated and the embryos then disinfested. The phytohormones 2,4-D, BA and GA3 induced callus on both the embryonic axis and cotyledons. To form callus on young leaves and petioles of the dwarf cultivars 'Xue Li Zi Yu' and 'Zi Xia Lin' 4.521JM 2.4-D and 4.44J.JM BA were required. Addition of 1.44J.JM GA3 to this medium was needed for cv. 'Cai Lan' to survive and ultimately form callus.

Chinese tree peony Paeonia suffruticosa Andr. comprises more than 500 cultivars. These attractive ornamentals have large, colourful flowers sought after in many countries. To maintain desirable hereditary traits the plants are propagated vegetatively. This process is time consuming and slow, contributing to the rarity and high cost of the plants (Aoki and Inoue 1992). As a result, many attempts have been made to micropropagate the plants, using different explants with varying success. Explants used include zygotic embryos (Demois and Partanen 1969), flower buds (Meyer 1976), stems (Gildow and Mitchell 1977) and roots (Meyer 1976, Gildow andMitchell 1977). Only callus formation was achieved. Plantlet regeneration was achieved from axillary buds, leaves and petioles of cv. 'Qing Long Wo Mo Chi' and #18 (Li eta/. 1984}, stems of cv. 'Papaveracea' (Harris and Mantell 1991 ), and buds of cv. 'Mme de Vatry' (Bouza et al. 1994). Little or no information is available on the elite dwarf cultivars and cv. 'Fengdanbai' which is a hardy plant.
In the case of herbaceous peonies P. anomala (Brukhin and Batygina 1994), P. a/bit/ora (Kim and Lee 1995), P. mlokosewitschii and P. tenuifo/ia (Orilikowska et a/. 1998) somatic embryogenesis has been achieved. There is no information with respect to P. suffruticosa. Before success with this technique can be achieved it is necessary to improve decontamination and minimise excessive culture browning. In addition, it is necessary to obtain an optimal medium containing the most efficient hormone supplements.
'Cai Lan', 'Xue Li Zi Yu' and 'Zi Xia Lin' were raised in a greenhouse. Upon sprouting, axillary buds, young expanding leaves, fully expanded leaves and active roots (3mm diameter) were used as explants. Seeds of cv. 'Fengdanbai' were collected from field grown plants after the follicles had ripened in autumn. The moisture content of the seed lot was 12% and the mass of 1 000 seeds was 238g. The seeds were stored at 5°C prior to use. Leaves were first rinsed in running tap water for 20min and then decontaminated in groups using three methods: 1.) Immersion into 70% (vlv) ethanol for 7-1 Osee and then into 0.5, 1.0 and 3.5% NaOCI (active ingredient in commercial bleach) for 15, 20 and 25min respectively; 2.) Immersion into 70% (v/v) ethanol for 7-1 Osee and then into 0.1% (wlv) HgCI~ for 3, 5 and ?min respectively; 3.) Immersion in a mix· ture of 20% (v/v) ethanol and 0.075% (w/v) HgCI~ for 5, 7 and 9min respectively. All explants were subsequently rinsed 3 times (3min each) in sterile distilled water. Tween 20 (1 drop per 50ml) was added to each sterilant. In the case of buds, scales were removed whereafter they were treated as the leaves. Roots were heated in water at 42°C for 2h (langens-Gerrits et a/. 1998) prior to decontamination. The procedures used were the same as for the leaves.
Seeds were decontaminated using three methods: 1.) Immersion in 70% (v/v) ethanol for 2min followed by 30min in 3.5% (active ingredient) NaOCI or 1Om in in 0.1% (w/v) HgCiz 2.) The seed coat was removed and the embryos were immersed in a mixture of 20% (v/v) ethanol and 0.075% (wlv) HgCb for ?min; 3.) A combination of methods 1 and 2. Contaminated seeds were removed after the intact seeds had been in culture for 60 days, the embryos excised and then decontaminated using method 2. The experiments were repeated twice with 25 replicates per treatment and the data subjected to statistical analysis (Steel et a/. 1997).
Three methods were used to reduce browning : 1.) Depending on explant vigour. young (emerged but unexpanded), expanding, fully expanded leaves, and the bud tips (less than 3mm in length) were excised under aseptic conditions and used ; 2.) Leaves and buds were soaked in 0.8mM ascorbic acid or 0.8mM citric acid (George and Sherrington 1984) or a combination of these acids (0.4mM each) for 20min prior to decontamination. Explants were then innoculated on to a medium containing 0.4mM of both chemicals; 3.) Leaves and buds were incubated on medium free of activated charcoal (AC) or containing 0.3% (w/v) AC respectively. Browning was recorded every 5 days for 35 days. Each treatment contained 25 replicates. For buds only 5 replicates were available.
Two media , Murashige and Skoog (1962) (MS) and the Woody Plant Medium (WPM) of Lloyd and McCown (1980). previously used fo r tree peonies, were employed. The pH of these were adjusted to 5.8 and they were solidified wi th Difco agar (0.8w/v). On WPM sig ns of a Ca-deficiency were observed. This was rectified by raising th e CaC I~ level of the medium to 6mM.
The disinfestation solution. explant and time applied all influenced the degree of decontamination recorded. While not always recommended (George and Sherrington 1984). the solutions con taining HgCb were more efficient in decontaminating explants. The highest percentage decontamination was obtained by immersion in the ethanoi/HgCiz mixture for ?min (Table 1 ). All seeds with intact testae were contaminated regardless of how harsh the treatment. Removal of such testae and re-decontaminating the embryos by immersion in the mixture of sterilants for ?min led to 86% decontamination and 45% germination within 30 days. This method was also most effective for the roots, yielding 33% decontaminated and viable explants after 60 days.
While the older explants turned brown on the media, the young leaves and buds remained green. Soaking in citric or ascorbic acid did not remedy this. Leaves placed on media containing 0.3% AC did not turn brown. The leaves, buds and embryos died on 1/2 MS and MS, survived on WPM, but subsequently developed characteristic signs of Ca deficiency. This was rectified by inclusion of 6mM CaCb to WPM. New green buds emerged and chlorosis disappeared within 15 days (Figure 1 ).
Addition of 2,4-D to the medium induced embryos of cv.  (Figure 20-G). The fou r hormone combinations used in an attempt to promote callus growth did not show any major differences in evoking callus formation over a period of 45 days (Figure 3). After this time, however, callus growth significantly improved on a med.ium containing  Following the original work of Li eta/. (1984) in vitro propagation of tree peonies has been studied frequently. At present, micropropagation does not yet offer a viable commercial option for propagation. Explant contamination and browning are still major problems (Buchheim and Meyer 1992). The present study indicated that by using a mixture of 20% ethanol and 0.075% HgCI2 decontamination can be improved compared to previous results (Gildow and Mitchell 1977, Harris and Mantell 1991, Bouza et at. 1994. This study also showed that vigorously growing material could be easily decontaminated and browning contained. The addition of 0.3% AC was effective in preventing tissue and media browning. In earlier studies (Li et a/. 1984, Harris and Mantel 1991, Bouza et at. 1994, lkuta et at. 1995 MS medium with a high nitrogen content was used. In our studies the dwarf cultivar material died on MS but survived on WPM. The overall growth was improved with the addition of 6mM CaCI2 to this medium. An increased need for higher Ca in the growth media of tre e peonies was highlighted earlier. Bouza et at. (1984) reported that doubling the Ca concentration resu lted in good multiplication rates.