Somatic embryogenesis and plant regeneration from immature seeds of Magnolia obovata Thunberg

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1 Plant Biotechnol Rep (2007) 1: DOI /s ORIGINAL ARTICLE Somatic embryogenesis and plant regeneration from immature seeds of Magnolia obovata Thunberg Yong Wook Kim Æ So Young Park Æ In Sun Park Æ Heung Kyu Moon Received: 3 August 2007 / Accepted: 17 September 2007 / Published online: 30 October 2007 Ó Korean Society for Plant Biotechnology and Springer 2007 Abstract We have tested plantlet formation by somatic embryogenesis using immature seeds of Magnolia obovata. Seed collection date appeared to be critical for embryogenic cell induction. The optimal collection date was 3 4 weeks postanthesis. The embryogenic cells proliferated, formed somatic embryos, and were subsequently converted into normal plantlets under optimized culture conditions. Somatic embryo formation from the embryogenic calli was better on sucrose medium than on glucose medium. The optimum level of sucrose appeared to be 3% with an average of 28 somatic embryos per plate. About 25% of somatic embryos were converted into normal plantlets in 1/ 2 MS medium containing gibberellic acid (GA 3 ). During somatic embryo germination, secondary embryogenesis was frequently observed in the lower part of the hypocotyl or radicle ends of germinating somatic embryos. Finally, about 85% of converted plantlets survived in an artificial soil mixture, were transferred to a nursery, and have grown normally. Keywords Japanese cucumber tree Immature zygotic embryos Plant conversion Y. W. Kim S. Y. Park H. K. Moon (&) Biotechnology Division, Korea Forest Research Institute, Suwon , South Korea jesusmhk@hanmail.net I. S. Park United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Saiwai-Cho, Fuchu-Shi, Tokyo , Japan Introduction Magnolia obovata is native to Japan and was introduced to Korea around the 1920s. The trees are deciduous, with gray bark, and grow up to 30 m. The tree has been planted as a garden tree because of its beautiful shape. The tree seeds ripen in September to October and most plants have been propagated from seedlings (Kim 1994). Magnolia species can be propagated by seeds, rooted cuttings, layering, grafting, and budding. Because magnolias have been the object of intense horticultural interest for centuries, methods for breeding and vegetative propagation of this group are well developed and have been well described (Merkle 1999 and papers cited therein). Micropropagation of Magnolia spp. is not sufficiently successful, however. A preliminary study of micropropagation of M. grandifolia using shoot-tip cuttings failed to produce plantlets (Tobe 1990). The first successful in-vitro propagation was achieved in M. x soulangiana (Maene and Debergh 1985; Kamenicka 1996). They found that exvitro rooting was improved by addition of water or water supplemented with IBA or sucrose to the cultures seven days before transplanting to peat potting mix. Kamenicka (1998) also tested the effect of various carbohydrate treatments on in-vitro shoot growth and rooting of the saucer magnolia. On the other hand, several results have suggested Magnolia spp. can be propagated by somatic embryogenesis using immature seeds. Intensive research has been done by Merkle s group and several papers have been published (Merkle and Watson-Pauley 1993, 1994; Merkle and Wiecko 1990; Merkle 1999). However, there has been no report of micropropagation of Magnolia obovata until now. This study was conducted to develop a micropropagation technique for the species using somatic embryogenesis.

2 238 Plant Biotechnol Rep (2007) 1: Materials and methods Explants and surface disinfection In 2003, developing fruits (aggregates of follicles) were collected, at about 10-day intervals, from 3 to 7 weeks postanthesis, from three trees growing on the campus of Seoul National University, Suwon, Korea. Individual seeds were dissected from the aggregates using a surgical knife and disinfested using the sequence: 70% ethanol, 30 s; 2% sodium hypochlorite 10 min; rinsing four times with sterile distilled water. Seeds were bisected longitudinally with a scalpel and the halves were placed cut, surface downward, on 25 ml semisolid callus induction medium (Table 1) in Petri dishes (87 15 mm). During explanting, no zygotic embryos could be observed inside the seeds under a dissecting microscope and endosperm, if present, was still in the liquid stage. Petri dishes were sealed with Parafilm and incubated in the dark at 25 ± 1 C. Explants were transferred to fresh medium 3 days later, then subcultured at monthly intervals. Callus and embryogenic callus induction To induce callus or embryogenic callus formation in initial cultures we prepared a callus-inducing medium consisting of MS medium (Murashige and Skoog 1962) that contained 3% sucrose (Sigma S5390, Grade I), 0.3% gelrite (Aldrich, USA), and which was supplemented with 1.0 mg L 1 2,4- D (2,4-dichlorophenoxyacetic acid), either alone or in combination with 0.01 mg L 1 TDZ (thidiazuron) or 1.0 g L-glutamine (Table 1). Glutamine was added after filter sterilization. The medium was adjusted to ph 5.8 with 0.1 mol L 1 NaOH or HCl before addition of gelrite. After autoclaving at 1.05 kg cm 2 and 121 C for 20 min, 25 ml medium was poured into each Petri dish (87 15 mm). The distribution of explants was five segments with five or Table 1 Normal callus induction, embryogenic callus induction, and zygotic embryo germination by the different culture dates and using different plant-growth regulators in Magnolia obovata Date of culture Medium and PGRs a (mg L 1 ) No. of explants cultured Normal callus induction (%) Embryogenic callus induction (%) Zygotic embryo germination (%) June 11 (1) MS + 2,4-D (2) MS + 2,4-D L-glutamine 1 g L (3) MS + 2,4-D TDZ (4) MS + 2,4-D TDZ L-glutamine 1 g L June 17 (1) MS + 2,4-D (2) MS + 2,4-D L-glutamine 1 g L (3) MS + 2,4-D TDZ (4) MS + 2,4-D TDZ L-glutamine 1 g L June 30 (1) MS + 2,4-D (2) MS + 2,4-D L-glutamine 1 g L (3) MS + 2,4-D TDZ (4) MS + 2,4-D TDZ L-glutamine 1 g L July 11 (1) MS + 2,4-D (2) MS + 2,4-D L-glutamine 1 g L (3) MS + 2,4-D TDZ (4) MS + 2,4-D TDZ L-glutamine 1 g L July 18 (1) MS + 2,4-D (2) MS + 2,4-D L-glutamine 1 g L (3) MS + 2,4-D TDZ (4) MS + 2,4-D TDZ L-glutamine 1 g L July 28 (1) MS + 2,4-D (2) MS + 2,4-D L-glutamine 1 g L (3) MS + 2,4-D TDZ (4) MS + 2,4-D TDZ L-glutamine 1 g L All culture medium contained 3% added sucrose and was gelled with 0.3% gelrite

3 Plant Biotechnol Rep (2007) 1: more replicates, depending on seed-collection date (Table 1). Following this inoculation, the cultures were maintained in a standard tissue culture room at 25 ± 1 C in darkness. After 6 weeks of culture PEDC (pre-embryogenic determined cell)-type somatic embryos were observed in some explants (Fig. 1a). To induce pre-embryogenic mass (PEMs), these somatic embryos were sliced about 1 mm thick using a surgical knife and sub-cultured in embryoinduction medium (EIM; MS medium with 1.0 mg L 1 2,4- D, 3% sucrose, and 0.3% gelrite). After 4 weeks in culture a friable aggregation of PEMs, yellow in color, was obtained (Fig. 1c). These PEMs were sub-cultured and proliferated on fresh EIM, in darkness, at 3-week culture intervals. Induction of somatic embryos Somatic embryos were induced from embryogenic calli on 1/2 MS medium supplemented with either sucrose (0, 1.0, 3.0, or 5.0%) or glucose (1.0, 2.0, 3.0, or 5.0%), which was gelled with 0.3% gelrite (Table 2). For plating, about 0.5 g embryogenic calli was placed in 100-mL Erlenmeyer flasks containing 30 ml 1/2 MS liquid medium supplemented with 2% sucrose without phytohormones. The cultures were agitated on a gyratory shaker at 120 rpm. One day later they were filtered with a cell-dissociation sieve (40 mesh, Sigma S0770); the floating matter was discarded and the cells were plated on to EIM after vacuum suction on filter paper. About 0.15 g of liquid medium containing cells was dispensed on each plate. Periodically, developing somatic embryos were examined under a dissecting microscope and the total number of early cotyledonary stage embryos was counted during 4 weeks of culture. Germination of somatic embryos and plantlet conversion Mature somatic embryos showing early cotyledonary stages with greening were transferred to 1/2 MS medium containing one of two different gelling agents (0.8% agar or 0.3% gelrite) to induce embryo germination and plantlet conversion. To promote embryo germination, 1.0 mg L 1 GA 3 was added to the medium (Figs. 2, 3). On the other hand, the frequency of secondary somatic embryo Fig. 1 Somatic embryogenesis and plant production via immature zygotic embryo cultures of Magnolia obovata. a PEDC-type somatic embryos induced from primary cultures ( 10); b Germinated zygotic embryo from explant without callus formation in primary culture medium ( 9); c Embryogenic callus (PEMs) showing friable texture and pale yellow color ( 10); d Normally induced somatic embryos at globular to torpedo stage ( 10); e Advanced somatic embryos from (d) at different stages of development ( 15); f Matured somatic embryos at early-cotyledonary stage ( 8); g Greened somatic embryos from (f), ready for germination ( 8); h Converted plantlets after germination (arrows show the secondary somatic embryos formed in lower part of the hypocotyls or root ends); i Field grown two-year-old plants derived from somatic embryos of M. obovata

4 240 Plant Biotechnol Rep (2007) 1: Table 2 Effect of sucrose and glucose on somatic embryo induction from embryogenic callus of Magnolia obovata Treatment (%) Sucrose ± ± ± 3.9 Glucose ± ± ± ± 1.9 No. of cotyledonary stage somatic embryo per plate The culture medium used was half-strength MS medium with different levels of sucrose or glucose Mean ± standard deviation % of normal conversion Fig. 2 Effect of medium on normal somatic embryo conversion. Medium 1 is 1/2 MS with 1.0 mg L 1 GA 3 and 0.8% agar. Medium 2 is 1/2 MS with 1.0 mg L 1 GA 3 and 0.3% gelrite % of secondary somatic embryo formation Fig. 3 Effect of medium on secondary somatic embryo formation. Medium 1 is 1/2 MS with 1.0 mg L 1 GA 3 and 0.8% agar. Medium 2 is 1/2 MS with 1.0 mg L 1 GA 3 and 0.3% gelrite formation was examined during the germination stage. Normally converted plantlets were transferred to plastic rectangular boxes ( cm) containing a mixture of artificial soil (peatmoss vermiculite perlite, 1:1:1) and cultivated in a greenhouse as described our earlier report (Moon et al. 2006). 2 Results and discussion Culture initiation and PEMs induction After three days of culture, most explants became brown and excreted dark compounds, probably phenolic compounds, into the medium. It was, therefore, necessary to sub-culture the cultures into fresh medium of the same composition. Generally, callus induction response was very poor, irrespective of seed-collection date. Non-embryogenic callus or embryogenic callus formation was not distinctly different among the four media (Table 1). Callus formation capable of embryogenesis was observed only when the seed-collection date was 11 or 17 June, otherwise either formation of non-embryogenic callus or direct germination of zygotic embryos was observed for most explants (Table 1; Fig. 1b). The two dates were approximately three to four weeks postanthesis and this appeared to be the time-window for induction of somatic embryogenesis in M. obovata (Table 1). The embryogenic callus was friable and yellow in color, contained globular stage embryos (Fig. 1c), and could be maintained in MS medium supplemented with 1.0 mg L 1 2,4-D, in darkness, by regular subculture. A similar result was also observed for M. virginiana (Merkle 1999). Otherwise, directly formed somatic embryos from explants were also observed in initial culture medium irrespective of treatments (Fig. 1a). These embryos appeared to be PEDC-type somatic embryos showing abnormality, and did not regenerate into plantlets via the normal embryo development process (data not shown). However, interestingly, about 10% of these embryos could form friable embryogenic callus when re-cultured on MS medium with 1.0 mg L 1 2,4-D. Therefore, this culture technique can be used as an alternative to induce embryogenic callus formation in M. obovata. Several previous results have shown that embryogenic callus formation, and its maintenance, for Magnolia species appears to differ greatly among species (Merkle 1999). Merkle (1999) concluded that 2,4-D treatment was necessary for PEM formation and proliferation in M. cordata, M. fraseri, and M. pyramidata, whereas the embryogenic callus of M. virginiana and M. macrophyllaas could be maintained in hormone-free medium for several years. In the current study, embryogenic callus of M. obovata needed 2,4-D to maintain embryogenic capacity, because the calli turned brown and died gradually in hormone-free medium. Somatic embryo induction When embryogenic cell suspensions were plated on EIM, globular-stage somatic embryos began to form after

5 Plant Biotechnol Rep (2007) 1: weeks of culture. Although most somatic embryos were observed within 3 weeks, irrespective of treatments, the numbers of somatic embryos differed among treatments (Table 2). Generally, sucrose was more effective than glucose for somatic embryo induction. The highest number of SEs was induced at the 3% level. Because these SEs formed at different times, they grew with different developmental stages (Fig. 1e). Sucrose is one of the most important carbon sources, and it has been used frequently in plant tissue culture work (Fuentes et al. 2000). In somatic embryogenesis it has been also used as a carbon and energy source, and at high concentrations it enhanced somatic embryo induction frequency caused by osmotic stress (Iraqi and Tremblay 2001). According to our recent work, somatic embryo germination and conversion was also greatly affected by various osmoticums and better results were obtained after sucrose treatment (Kim et al. 2005). In the current study it is difficult to reach a firm conclusion, because we compared two carbon sources only; nevertheless, sucrose appeared to be better for somatic embryo induction in M. obovata. In most somatic embryogenesis studies of several Magnolia species sucrose has been used more frequently (Merkle 1999). Germination and plantlet conversion Embryo germination was defined as embryos showing hypocotyl elongation and root development, whereas plantlet conversion was considered as embryos showing normally developed cotyledons and epicotyls after germination. Using early-cotyledonary-stage SEs, embryo germination was performed on 1/2 MS medium with 1.0 mg L 1 GA 3 and two different gelling agents (Fig. 2). There was no difference between embryo germination and conversion in the two different gelling agents the conversion rate was 25 and 23% in 0.8% agar and 0.3% gelrite, respectively. In a similar result, about 25% conversion rate was reported for sweetbay magnolia (Merkle and Wiecko 1990). On the other hand, less than 10% conversion rate of somatic embryos was reported for Fraser magnolia and yellow cucumber tree (Merkle and Wiecko 1990). These results suggest that somatic embryo germination and conversion in Magnolia species is still not efficient. It appeared that the lower conversion rate of SEs was caused by morphological abnormality of SEs, including single-cotyledon embryos (horn type), fused cotyledons, and/or embryos with root only. Similar results have been reported for many woody plant species (Merkle 1997; Kim et al. 2005; Moon et al. 2005, 2006). It is, therefore, necessary to develop more efficient techniques to improve the conversion rate for M. obovata. On the other hand, about Fig. 4 Normally grown 4-year-old plants derived from somatic embryos of Magnolia obovata 60% of germinating embryos formed secondary somatic embryos at the lower portion of the hypocotyl or root ends (Fig. 1h). These secondary somatic embryos were slightly more abundant in gelrite medium (Fig. 3). Similar results were also observed for radicle ends of the sweet magnolia (Merkle and Wiecko 1990). These secondary somatic embryos, which are capable of successive embryogenesis under optimized culture conditions, are being studied to establish repetitive somatic embryogenesis in M. obovata. In conclusion, we have produced plantlets via somatic embryogenesis using immature seeds of M. ovobata. Although the frequency of embryogenic callus formation was very low, the calli were proliferated easily by successive sub-culture on to fresh medium. One difficulty was abnormal shapes of somatic embryos, which resulted in poor germination and conversion to plantlets. Therefore, more study in this area is needed. Fortunately, the converted plantlets survived well (about 85%) in potting mixture and have grown (mean height ± 26.8 cm) normally for up to four years without morphological abnormalities (Fig. 4). The results suggest this technique may be applicable to micropropagation of M. obovata. Acknowledgments This work was supported in part by grant No. FG from the Korea Forest Research Institute. References Fuentes SRL, Calheiros MBP, Manetti-Filho J, Vieira LGE (2000) The effects of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora. Plant Cell Tissue Organ Cult 60:5 13 Iraqi D, Tremblay FM (2001) The role of sucrose during maturation of black spruce (Picea mariana) and white spruce (Picea glauca) somatic embryos. Physiol Plant 111: Kamenicka A (1996) Rooting of Magnolia x soulanglana microcuttings. Biologia 51:

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