04. F13-04.pdf459.7KB

Introduction

 Peonies are perennial ornamental plants of the genus Paeonia, which belongs to the family Paeoniaceae and consists of 33 species (Rogers 1995). These species are divided into herbaceous and tree peonies. Peonies are grown successfully in moderate, cold-winter climatic zones, where they bloom in May-July (Barzilay et al. 2002). Herbaceous peonies are valued as medicinal and ornamental plants in Korea (Sohn et al. 2002). They are also famous garden and cut flower plant, and can also be used as potted plants. Herbaceous peony is generally propagated by seed or asexually, including by plant division, cottage, grafting and layering. However, the rate of vegetative propagation is very slow and seed germination in nature is also very poor due to complex sequential dormancy of seed. Thus complex stratification is required to induce the germination of seeds. Embryo culture can help to overcome embryo non-viability, seed dormancy and related problems (Stanys et al. 2007).

 Clonal propagations of P. lactiflora have been reported through the shoot tips (Hosoki et al. 1989) and underground buds (Wu et al. 2011; Yu et al. 2012). In vitro seed germination in Paeonia lactiflora has been reported earlier with cytokinin enriched MS medium and two-month cold stratification (Stanys et al. 2007). They reported that supplementation of BA and IAA growth regulators to a nutrition medium, may partially replace stratification. Epicotyl dormancy was broken in P. lactiflora 'Pink Dawn' embryos cultured on medium containing 1 or 10 μM BA. A highly significant increase in leaf number occurred when embryos were cultured on modified LS medium containing BA and GA₃ (Buchheim et al. 1994).

 Clonal propagation through somatic embryogenesis has become an essential method for the improvement of many economically important plants (Samson et al. 2006). Direct embryogenesis reduces the time required for plant propagation, which may be beneficial in minimizing cultureinduced genetic changes and/or variations (Sivanesan et al. 2011a). Somatic embryogenesis offers an excellent experimental system to study the physiological and biochemical aspects of embryo development (Shohael et al. 2007; Sivanesan et al. 2011b). The process of somatic embryo development on initially formed or primary somatic embryo is termed as secondary somatic embryogenesis. It has great potential for large-scale micropropagation and it can be utilized for genetic transformation. Further, embryogenicity can be maintained for a long period of time by repeated cycles of secondary embryogenesis. It is also reported that efficiency of explants in secondary embryogenesis is higher than in primary embryogenesis (Kim et al. 2012).

 Only a few reports are available on somatic embryogenesis of peony. Somatic embryos were induced from anthers and cotyledons of P. lactiflora (Kim and Lee 1996; Kim et al. 2006; Lee et al. 1992). However, till date there are no reports available on secondary somatic embryogenesis of Paeonia species. The objectives of the present study were to evaluate the effect of BA and GA₃ on zygotic embryo germination and to determine the effect of plant growth regulators (PGRs) on somatic embryogenesis, using cotyledons of P. lactiflora as the explants.

Materials and Methods

 Seeds were collected from the mature, field-grown plants (Fig. 1A), washed thoroughly under running tap water with few drops of Tween 20 for 30 min, and then washed with distilled water. Seeds were treated with a 70% (v/v) ethanol (Yakuri Pure Chemicals, Kyoto, Japan) for 5 min, 1.5% (v/v) sodium hypochlorite (NaOCl) (Yakuri Pure Chemicals, Kyoto, Japan) for 5 min followed by 3-4 washes with distilled water. Above steps were repeated again under a laminar air flow for surface sterilization.

Fig 1. (A) Seeds of P. lactiflora; (B) excised zygotic embryos from the seeds; and (C) germinated embryos 30 days after inoculation.

 The culture medium consisted of Murashige and Skoog (MS) salts and vitamins (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose, and solidified with 0.8% (w/v) agar (Plant Agar, Duchefa Biochemie, The Netherlands). The pH of the medium was adjusted to 5.80 using 0.1 N NaOH or 0.1 N HCl before autoclaving at 121^°C for 15 min. Plant growth regulators (PGRs) were added to the basal medium prior to pH adjustment and sterilization. The gibberellic acid (GA₃) was filter sterilized and added to the autoclaved medium. Other PGRs were added to the medium prior to pH adjustment and disinfection. All cultures were maintained at 25 ± 2^°C under a 16 h photoperiod with 30 μmol m^-2s^-1PPFD provided by cool white fluorescent light (TL-D 32W/865 RS 1SL, Philips, The Netherlands).

 Zygotic embryos (Fig. 1B) excised from seeds were placed in 9 cm Petri dish (20 embryos per Petri dish) containing 20 mL of the MS medium supplemented with 0.1% (w/v) activated charcoal (AC) (Sigma-Aldrich, St. Louis, MO, USA), different concentrations and combinations of N⁶ benzyl-adenine (BA), and GA₃ (Table 1). Germination was recorded after 30 days of inoculation. Cotyledon explants (0.5 mm in length) isolated from the 30 days old germinated embryos were cultured on the MS medium supplemented with various combinations of BA and 2,4- dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA), and GA₃ combinations. Observations were made at a 30 days interval. The frequency of somatic embryo formation was determined by counting explants forming somatic embryos from the total number of the cultured explants after 90 days of culture.

 To obtain the secondary embryos, torpedo stage primary somatic embryos were transferred to MS medium supplemented with 3.0 mg · L^-1 BA, 1.0 mg · L^-1 NAA, and 1.0 mg · L^-1 GA₃. The frequency of SSE formation was determined by counting embryos forming SSEs from the total number of the cultured embryos after 45 days of culture.

 For somatic embryo conversion, the torpedo stage embryos were separated from the explants and cultured on the MS medium supplemented with BA and GA₃ each at 1.0mg · L^-1 for conversion. Somatic embryos were subcultured at 4 weeks interval on the same culture medium. Embryo germination was calculated after 30 days as the percentage of number of germinated embryos per total number of somatic embryos.

 In each treatment, about 25 zygotic embryos, cotyledons or somatic embryos were used and the experiment was repeated thrice. Data were statistically analyzed by analysis of variance (ANOVA) followed by Duncan multiple range test at 5% probability level. Data analysis was performed using SAS computer package (SAS Institute Inc., Cary, NC, USA).

Results and Discussion

 The surface sterilization method helped to produce 100% aseptic cultures. Various combinations and concentrations of BA and GA₃ were tested for zygotic embryo germination. Although germination occurred when embryos were cultured on the MS medium supplemented with BA, and GA₃, the frequency of germination varied among treatments (Table 1). In contrast, embryos failed to germinate on the PGR-free MS medium. Due to complex sequential dormancy of peony seeds it usually takes 2 years for germination under natural conditions (Griess and Meyer 1976; Tian et al. 2010). Asynchronic development of different embryo parts and prolonged germination of seeds are characteristic of this genus (Stanys et al. 2007). Plant growth regulators are found to play an important role in seed germination. The stimulating effects of cytokinins on seed germination have been reported in many plants (Guleryuz et al. 2011; Kabar 1998). It has been also reported that GA₃ is extremely important for seed germination (Finkelstein 2004; Koornneef et al. 2002). Gibberellins are known as growth-promoting hormones, being involved in several processes during plant development, such as shoot growth, flower development, dormancy release, and seed germination (Linkies and Leubner-Metzger 2012). In the present study, the greatest germination percentage (95%) was observed when the MS medium was supplemented with 1.0 mg · L^-1 BA, and 0.5 mg · L^-1GA₃ (Table 1). The medium supplemented with these two PGRs resulted in embryo germination with demarcated cotyledonary leaves as recorded after 30 days (Fig. 1C). The number of plants raised by this embryo germination may be greater than growth from seeds in nature and the time duration will also be less.

Table 1. Effects of BA and GA₃ on germination of zygotic embryos of P. lactiflora.

Table 2. Effects of BA and 2,4-D on responses of cotyledon explants of P. lactiflora.

Table 3. Effects of PGRs and their concentrations of on somatic embryo induction in P. lactiflora measured at 90 days after inoculation.

Fig 2. (A) Somatic embryos were induced from cotyledon explants cultured on MS medium with 3.0 mg · L^-1 BA, 1.0 mg · L^-1 NAA, and 1.0 mg · L^-1 GA₃ after 60 days, arrow showing globular embryos and (B), after 90 days, arrow showing torpedo-shaped embryos.

Fig 3. Secondary somatic embryogenesis observed on primary somatic embryo, when the latter was transferred onto the MS medium supplemented with 3.0 mg · L^-1 BA, 1.0 mg · L^-1 NAA, and 1.0 mg · L^-1 GA₃. Arrow showing secondary somatic embryos (SSE), Primary somatic embryo (PSE) having cotyledon (C), and root (R).

Fig 4. Conversion of somatic embryo cultured on the MS medium supplemented with 1.0 mg · L^-1 BA and 1.0 mg · L^-1 GA₃ (A) after 10 days, and after 60 days (B).

 Swollen explants and callus were observed after 30 days when cotyledon explants were cultured on the MS medium supplemented with different combinations of BA and 2,4-D. Greenish-white calli were observed when these swollen cotyledons were further subcultured on the same medium for 90 days (Table 2). Compact green callus was obtained when concentrations of BA and 2,4-D were increased equally. Kim and Lee (1996) reported somatic embryos were formed directly on the surface of cotyledon explants of Paeonia albiflora cultured on the MS medium supplemented with BA and 2,4-D. However, in the present study somatic embryos were not formed in the presence of BA and 2,4-D.

 When cotyledons were cultured on the medium with combinations of BA, NAA, and GA₃, somatic embryos were observed after 60 days; however data was recorded after 90 days (Table 3). Kim et al. (2006) reported direct somatic embryos formed on the MS medium supplemented with 1.0 mg · L^-1 abscisic acid (ABA) from cotyledon explant and with 2.0 mg · L^-1 phenyl acetic acid (PAA) from the anther explant of peony after 90 days of culture under complete darkness. The embryos were loosely attached to the surface of the explant and they were white or green in color, small and globular in shape appearing in clusters (Fig. 2A). In the present study, the highest frequency of somatic embryo induction (72.5%) was obtained on the MS medium supplemented with 3.0 mg · L^-1 BA, 1.0 mg · L^-1 NAA, and 1.0 mg · L^-1 GA₃, with a mean number of 14 embryos per explant (Table 3). To induce somatic embryogenesis, a balance of auxin and cytokinin is important, and BA is one of the cytokinins commonly used in combination with other plant growth regulators (Gaj 2004; Rajabpoor et al. 2007). Similarly, somatic embryo induction and maturation on the MS medium containing BA and NAA was observed in Digitalis lamarckii (Verma et al. 2011), P. albiflora (Kim and Lee 1996), Psoralea corylifolia (Sahrawat and Chand 2001), and Solanum surattense (Rama Swamy et al. 2005).

 In the present study, various stages of globular- heart, and torpedo- shaped embryos could be observed simultaneously on the same medium (Fig. 2B). All the embryos were morphologically normal. When primary somatic embryo was cultured on the MS medium with 3.0 mg · L^-1 BA, 1.0 mg · L^-1 NAA, and 1.0 mg · L^-1 GA₃ secondary somatic embryos (87%) were observed on the surface of primary somatic embryos (Fig. 3). Kim et al. (2012) reported in Panax ginseng that secondary embryogenesis mostly occurs from the regions such as hypocotyls and root pole. Similarly in the present results secondary somatic embryos have been observed from the root part of primary somatic embryo. Secondary somatic embryos have also been reported in Crocus vernus (Sivanesan et al. 2012) and Morus alba (Agarwal et al. 2004).

 When the torpedo stage embryos (Fig. 4A) were transferred to the MS medium supplemented with 0.1% AC, BA and GA₃ each at 1.0 mg · L^-1, they germinated with the shoot and root system (Fig. 4B). The somatic embryo germination was 100%. The inclusion of AC to the culture medium has been proven advantageous for embryo growth and normal development in several plant species (Dhekney et al. 2011; Sivanesan et al. 2011a; Thomas 2008). The maturation and conversion of somatic embryos into plantlets are significant steps during somatic embryogenesis. Our findings also reveal that plant growth regulators supported maturation and conversion of somatic embryos. It has been reported that BA and GA₃ promote embryo conversion in many plants such as Crocus vernus (Sivanesan et al. 2011b), Cnidium officinale (Lee et al. 2009), P. albiflora (Kim and Lee 1996), and Quercus rubra (Vengadesan and Pijut 2009). This proposed method of somatic embryo induction and germination will hasten germination from up to two years in nature, to only few months in vitro. The total period of time required for a peony plant to mature may be reduced by years.

 To summarize, the method developed is simple, reproducible, and efficient protocol for somatic embryogenesis from cotyledonary explant of P. lactiflora. Somatic embryogenesis offers an alternative and efficient means for plant multiplication. Since the plantlets developed directly without any intervening callus phase, somaclonal variations in the regenerated plants can be avoided.

Acknowledgements

 “This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. Project No: 109096-5”.