Plant Cell Reports (2000) 19 : 946–953

Q Springer-Verlag 2000

CELL BIOLOGY AND MORPHOGENESIS K. Tanaka 7 Y. Kanno 7 S. Kudo 7 M. Suzuki

Somatic embryogenesis and plant regeneration in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura)

Received: 30 April 1999 / Revision received: 7 October 1999 / Accepted: 27 January 2000

Abstract Somatic embryogenesis was observed in rayfloret explants of Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu on Murashige and Skoog medium containing high concentrations of 3indoleacetic acid (IAA) and kinetin. 1-Naphthaleneacetic acid also induced somatic embryogenesis but indole-3-butyric acid or 2,4-dichlorophenoxy acetic acid did not. Other cytokinins, such as 6-benzylaminopurine (BAP) and thidiazuron, were also not effective. No embryos were seen at lower IAA concentrations with kinetin and various concentrations of BAP, although higher BAP concentrations yielded many adventitious shoots. In contrast, no somatic embryogenesis was observed from leaves using any combination of plant growth regulators. Histologically, primordia showed a typical embryo shape with a well-developed vascular bundle between the shoot and the root primordia. Embryos had both stomata cells and a root system with polarity. Plants were efficiently regenerated from ray floret-derived embryos subcultured in the appropriate medium. Key words Chrysanthemum 7 Dendranthema grandiflorum (Ramat.) Kitamura 7 Plant regeneration 7 Somatic embryogenesis Abbreviations ABA: Abscisic acid 7 BAP: 6-Benzylaminopurine 7 2,4-D: 2,4-Dichlorophenoxyacetic acid 7 GA3: Gibberellic acid 7 IAA: 3-Indoleacetic acid 7 IBA: Indole-3-butyric acid 7 MS: Murashige and Skoog medium 7 NAA: 1-Naphthaleneacetic acid 7 TDZ: Thidiazuron Communicated by F. Sato K. Tanaka 7 Y. Kanno 7 S. Kudo 7 M. Suzuki (Y) Aomori Green BioCenter, 221–10, Yamaguchi, Nogi, Aomori 030–0142, Japan e-mail:msuzuki6mm.neweb.ne.jp Fax: c81-17-728-1017

Introduction Somatic embryogenesis has proved to be useful for micropropagation and the production of mutants, artificial seeds, and materials for use plant-genetic engineering (Pavingerova et al. 1994). Somatic embryogenesis generally occurs through two different pathways; namely, directly without forming the callus phase and indirectly following callus formation from explants. Direct embryogenesis, ultimately from single cells taken directly from explant tissue, may help to retain clonal fidelity. In spite of many attempts, however, it has been very difficult to induce either type of somatic embryogenesis in chrysanthemum. Whereas adventitious shoot regeneration from leaves and stems has already been reported in chrysanthemum (Roest and Bokelmann 1975), there have been only limited reports on the induction of embryos and embryoid-like structures (May and Trigiano 1991; Pavingerova et al. 1994; Shinoyama et al. 1997). May and Trigiano (1991) and Pavingerova et al. (1994) both reported obtaining embryogenesis from leaves of chrysanthemum; however, the shapes of the embryos reported by May and Trigiano (1991) were abnormal in appearance and the report by Pavingerova et al. (1994) lacked histological evidence. Recently, Shinoyama et al. (1997) reported the appearance of embryoid-like structures from the leaves of chrysanthemum (cv. Shuuho no tikara). However, shoot formation was observed subsequent to the root system being fully developed, and these authors could not obtain incontrovertible proof of embryogenesis, as was also the case with the previous reports. No further information exists on somatic embryogenesis in other chrysanthemum cultivars. Regeneration from petals of chrysanthemum has been achieved (Bush et al. 1976) and utilized for the production of new varieties via somaclonal variation. In the course of our study for producing somaclonal varients from petals, we occasionally observed somatic embryogenesis and plant regeneration from ray-florets explants of Dendran-

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thema grandiflorum (Ramat.) Kitamura (cv. Aboukyu). We report here our investigation of various combinations of plant growth regulators and optimization of the conditions for inducing embryogenesis from these rayfloret explants.

Materials and methods

matization of the plants was carried out with the box being wrapped with Saran Wrap (Asahi Chemicals, Tokyo, Japan), with about 10 pots in each box. After cultivation for 2 weeks, small holes were made in the wrap to enable an exchange of air and humidity. The plants were cultivated for 2 more weeks consecutively. Thereafter, the Saran Wrap was removed, and the plants were cultivated for another few weeks. After acclimatization, plants (about 12 cm tall) were transferred to pots containing soil and cultivated in a greenhouse. Scanning electron microscopy (SEM)

Plant material Dendranthema grandiflorum (Ramat.) Kitamura (cv. Aboukyu) was obtained from the Hachinohe Agricultural Center (Hachinohe-city, Aomori, Japan) and cultivated in a greenhouse and at a farm. At the farm, flowers bloomed annually, in October. Young leaves and ray florets of fully opened capitulums were used as material for organogenesis.

About 25-mm 2 samples were cut off and embedded quickly on specimen stubs, which were pre-frozen in liquid nitrogen for 3 min. The samples were then put into a specimen chamber and observed under freeze-dry conditions with a scanning electron microscope (Japan Electrics, JSM-5800LV). Histological sections

Induction of embryogenesis The ray florets and young leaves of chrysanthemum were surfacesterilized with 0.1% sodium hypochlorite and then rinsed four to five times with sterile distilled water. After sterilization, ovaries were excised from the ray florets using a disposable knife and the ray florets were then cut into 1-cm-long segments. Usually, we placed the abaxial sides of the excised ray florets on the surface of MS medium (Murashige and Skoog 1962) supplemented with various combinations and concentrations of plant growth regulators, such as IAA, NAA, BAP, kinetin and TDZ, and with 3% sucrose and 0.7% agar (pH 5.7). Plant growth regulators were added to the medium before autoclaving. These ray florets and leaves were cultured in the dark at 25 7C for 1 week, followed by a 1-month culture under a photoperiod of 12-h light (3000 lux) and 12-h dark Plant regeneration Plantlets were developed from somatic embryos by subculturing on growth regulator-free MS medium containing 2% sucrose and 0.7% agar in a Magenta box with filter (Life Technology, USA). Agar was carefully washed from the regenerated plants prior to their transfer to pots containing vermiculite and soil (1 : 2). Accli-

Embryos induced after 50 days of culture were embedded in 5% agar melted at 40 7C and cooled at 4 7C for fixation. Samples were sliced into 60-mm-thick sections by a Dohan Microslicer DTK3000 (Dohan, Japan) and observed in a light microscope.

Results and discussion Effects of plant growth regulators on organogenesis from leaf explants Leaves of Dendranthema grandiflorum (Ramat.) Kitamura (cv. Aboukyu) were cultured with various combinations and concentrations of plant growth regulators (Table 1). Initially 15 different conditions were studied with respect to the concentrations of IAA, NAA, kinetin and BAP. Among these concentrations (5.371 mM NAA, 5.708 and 57.08 mM IAA, 0.465 mM kinetin and 0.444–44.4 mM BAP), efficient callus formation was observed with 57.08 mM IAA, 0.465 mM kinetin and 2.22 or 44.4 mM BAP, and adventitious

Table 1 Response of leaf explants of Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu to various levels of plant growth regulators in MS medium containing 0.46 mM kinetin and supplemented with 3% sucrose and 0.7% agar after 38 days IAA (mM)

5.71 5.71 5.71 5.71 5.71 57.08 57.08 57.08 57.08 57.08 0 0 0 0 0 a

NAA (mM)

0 0 0 0 0 0 0 0 0 0 5.37 5.37 5.37 5.37 5.37

BAP (mM)

0 0.04 0.44 4.44 44.40 0 0.04 0.44 4.44 44.40 0 0.04 0.44 4.44 44.40

Numbers of leaves tested 20 20 19 20 19 20 5 16 20 20 20 20 16 20 20

Frequency (%) a of leaves showing Adventitous roots

Calli

85 a,b 45 c 37 c 0 0 100 a 100 a,b 75 b,c 0 0 100 a 95 a,b 50 c 0 0

15 e,f,g 55 c,d 42 d,e,f 75 b,c 32 d,e,f 0 0 25 d,e,f,g 100 a 80 b,c 0 5g 50 c,d,e 100 a 95 a,b

Means followed by the same letter are not significantly different according to the Waller-Duncan K-ratio t-test (Kp100, Pp0.05)

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root formation was observed frequently with 5.371 mM NAA and 8.88 or 22.2 mM BAP. May and Trigiano (1991) observed the formation of somatic embryos from leaves of chrysanthemum using a combination of 1.0 mg/l (4.52 mM) 2,4-D and 0.2 mg/l (0.89 mM) BAP. However adventitious shoot formation or somatic embryogenesis occurred with any combination and concentration of the growth regulators. The addition of GA3 (gibberellic acid) increased adventitious root formation (data not shown). There were no significant difference between full- and half-strength MS medium with respect to callus and adventitious root formation (data not shown). To investigate which side of the leaf explants was better for organogenesis, we placed leaves upside down or downside down on the medium. We observed no differences in callus and adventitious root formation between the two sides. Effects of plant growth regulators on embryo induction from ray-floret explants In chrysanthemum, the petals are another candidate as a source for organogenesis and plant regeneration, in addition to leaves and stems (Bush et al. 1976). Ray florets of Dendranthema grandiflorum (Ramat.) Kitamura (cv. Aboukyu) were cultured with various combinations and concentrations of plant growth regulators. The frequency of organ differentiation was scored after 38 days. We first compared the effects of auxins (Table 2). Ray florets were found to be the most responsive explant for embryogenesis. Somatic embryogenesis was observed upon the addition of high concentrations of IAA (57.08 mM) or NAA (57.08 mM) in the presence of 0.465 mM kinetin. IAA was especially able to induce embryogenesis at a high frequency (58%). In contrast, IBA and 2,4-D had no effect on inducing embryogenesis. As for the site of embryogenesis on a ray floret, the embryoid positions seemed to be independent of polarity since embryogenesis occurred both in the center of a ray floret as well as in the peripheral regions. Thus, embryogenic tissues were often very disarrayed. Cytokinins have been reported to induce somatic embryogenesis in Trifolium spp., flax, pea, Brassica napus, Brahmi (Tiwari et al. 1998 and references therein) solely, or in combination with an auxin (Zhong

et al. 1991; Neuenschwander and Baumann 1992; Samantaray et al. 1997). In preliminary experiments, we found that kinetin had the ability to induce embryogenesis. Consequently, we investigated the optimal concentration of kinetin for inducing embryogenesis (Table 3). In the presence of 57.08 mM IAA, 0.465 mM kinetin produced the highest frequency of somatic embryogenesis. No embryogenesis was observed in the absence of kinetin. Although May and Trigiano 1991) reported to have induced embryos from the leaves of chrysanthemum with the combination of 4.52 mM 2,4-D and 0.89 mM BAP, we could not induce any embryos from ray florets with this combination, as was the case with leaves. We could not explain clearly the different results because they did not refer to other combination of plant growth regulators. As the genotypes of chrysanthemum used in their study were different from those we used, the response to plant growth regulators might be also different. In the presence of 0.465 mM kinetin, BAP decreased the frequency of embryogenesis (Table 3). The frequency of somatic embryo formation was 56% in BAP-free medium, which was higher than that in the presence of BAP at any concentration studied here. When concentrations of BAP higher than 4.44 mM were added, the frequency of somatic embryogenesis declined, and more adventitious shoots were formed. Under such conditions, the induction of adventitious shoot primordia was observed over the entire embryoid surface. In contrast to results with high concentrations of BAP, calli and adventitious roots were formed from ray-floret explants with low concentrations of BAP (0–0.444 mM) in combination with 5.708 or 57.08 mM IAA. BAP was shown to inhibit embryogenesis in the case of cv. Aboukyu. Kintzios and Michaelakis (1999) also reported that fully developed, cotyledonary-stage somatic embryo from flower segments of chamomile could be induced only with the combination NAA and kinetin, although the formation of globular-stage somatic embryos was obtained with BAP instead of kinetin. Using the combination of 57.08 mM IAA and 0.46 mM kinetin, we observed somatic embryogenesis in 58% of ray florets (Table 2), that were first cultured in the dark for 1 week followed by culture under a photoperiod of 12-h light and 12-h dark for 1 month. May and Trigiano (1991) reported that 4.1% of leaves showed embryogenesis; these were cultured in the dark

Table 2 Response of ray-floret explants of Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu to various kinds of auxin in MS medium containing 0.46 mM kinetin and supplemented with 3% sucrose and 0.7% agar after 38 days Auxins

Concentrations (mM)

Numbers of ray florets tested

Frequency a (%) of ray florets showing Somatic embryogenesis

IAA IBA 2,4-D NAA a

57.08 57.08 57.08 57.08

249 30 30 30

a

58 0 0 17 b

Adventitious shoots

Adventitious roots

0 0 0 0

42 b 23 c 60 b 83 a

Means followed by the same letter are not significantly different according to the Waller-Duncan K-ratio t-test (Kp100, Pp0.05)

949 Table 3 Response of ray-floret explants ofDendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu to a combination of IAA and cytokinins in MS medium supplemented with 3% sucrose and 0.7% agar after 38 days IAA (mM)

0 0 57.08 57.08 57.08 57.08 57.08 5.71 5.71 5.71 5.71 5.71 57.08 57.08 57.08 57.08 57.08 a

Kinetin (mM)

0 0.46 0 0.46 2.32 4.65 23.24 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46

BAP (mM)

– – – – – – – 0 0.04 0.44 4.44 44.40 0 0.04 0.44 4.44 44.40

Numbers of ray florets tested

16 5 14 14 10 15 13 30 60 10 20 30 80 70 70 70 40

Frequency a (%) of ray florets showing Somatic embryogenesis

Adventitious shoots and roots

Adventitious roots

0 0 0 43 a 20 a,b 0 8b 0 0 0 0 0 56 a 44 a 46 a 23 b 0

0 0 0 0 0 27 a 8b 0 2d 10 c,d 50 b 80 a 0 0 0 11 c 58 b

0 0 0 0 0 7a 31 a 27 a,b,c 12 c 0 0 0 35 a,b 46 a 24 b,c 0 0

Means followed by the same letter are not significantly different according to the Waller-Duncan K-ratio t-test (Kp100, Pp0.05)

for 1 week followed by culture in 10 days of light and 35 days of darkness. TDZ is also known to induce multiple shoot formation in a broad range of plant species (Malik and Saxena 1992a, 1992b; Saito and Suzuki 1999) and somatic embryogenesis in many plants (Gill and Saxena 1993; Gill et al. 1993; Huetteman and Preece 1993; Lu 1993; Murthy et al. 1995; Murthy and Saxena 1998; Neuman et al. 1993; Sreenivasu et al. 1998; Visser et al. 1992). In the range from 0.45 to 45.4 mM TDZ, embryos, adventitious shoots and adventitious roots were not formed. Only callus formation was observed at a 9.1 mM concentration (data not shown). On the other hand, since ABA has been reported to promote embryo development (Bespalhok and Hattori 1998; Wakhul and Sharma 1998), we investigated the effect of ABA on mature embryo formation of cv. Aboukyu. In our studies, however, ABA showed no promotive effect for occurrence and development of somatic embryogenesis in cv. Aboukyu at the concentrations ranging from 0.38 mM to 3.8 mM (data not shown). Generally, there are two alternative ways to induce embryogenesis; namely, direct embryogenesis from the ray-floret surface and embryogenesis via calli. Both types of somatic embryogenesis were observed in this study (Fig. 1A,B). Direct embryogenesis from tissue sections, without the callus phase, seemed more suitable for mass propagation because genetic rearrangement was limited compared with the embryogenesis via calli, which often showed aberrent chromosome numbers during culture. We investigated the frequency of both types of embryogenesis. In 124 embryos formed from ray florets in a typical experiment, 20 embryos occurred via direct embryogenesis, while 104 embryos from embryogenesis via calli. Both types of embryos

formed on the ray-floret surface developed on plant growth regulator-free MS medium (Fig. 1D) and formed whole plants (Fig. 1E). Histological analysis Many types of embryoids appeared on the surface of ray florets. Among them, abnormally shaped embryoids were observed occasionally (Fig. 1B, right). To confirm the appearance of embryo-like structures in greater detail, we observed the structures microscopically by scanning electron microscopy. These structures revealed typical torpedo-shaped and, then, cotyledonary-stage somatic embryos (Fig. 2D). At first, embryo-like cell clusters appeared from surface cells (Fig. 2A) and grew into globular-stage somatic embryos (Fig. 2B). Leaf primordia were formed within approximately 10 days, (Fig. 2C), and the cotyledon was observed (Fig. 2D) after approximately 4 weeks. When cotyledonary-stage cells were observed by scanning electron microscopy, stomata and typical root-shaped cells were clearly present with opposite polarity (Fig. 2E,F). Longitudial sections of somatic embryos were observed microscopically (Fig. 3). A embryo at the early cotyledonary stage showed a central vascular bundle between a shoot primordium and a root primordium (Fig. 3A), and a well-developed vascular bundle was observed in a embryo at middle cotyledonary stage (Fig. 3B). On the other hand, no vascular connection was observed between a somatic embryo and surface cells of a ray-floret tissue (Fig. 3C). These results indicate that the regenerants were properly derived from genuine somatic embryos.

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O Fig. 1A–E Plant regeneration via embryogenesis from petals of Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu. A Globular-stage somatic embryos directly formed on the surface of petals, B embryos formed via calli derived from petal cells, C development of somatic embryos, D plantlets cultured on growth regulator-free MS medium containing 2% sucrose and 0.7% agar, that had developed from somatic embryos, E regenerated whole plants of chrysanthemum from somatic embryogenesis after approximately 8 weeks

M Fig. 2A–F Scanning electron microscopy of somatic embryos of the embryo-like cell cluster formed on the surface of petals. A Globular-stage somatic embryos developed from surface cells, B leaf primordia developed from A, C–D cotyledon formed on the surface of petals, E stomata cells on leaf parts of cotyledon, F typical root cells on root parts of cotyledon

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Conclusion We obtained many plants via somatic embryogenesis from ray-floret tissues. The protocol of embryogenesis developed here is simple and reproducible. Acknowledgements We thank Prof. S. Fukai (Kagawa University) for his kind discussion and Mr. K. Higuchi in Aomori Green BioCenter for encouragement.

References

Fig. 3A–C Histological sections of somatic embryogenesis in Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu. A Section of a somatic embryo at the early cotyledonary stage showing a central vascular bundle. R Root primordium, S shoot primordium. Bar: 500 mm. B Section of a somatic embryo at the middle cotyledonary stage showing the shoot and the root primordium. R Root primordium, S shoot primordium. Bar: 500 mm. C Tangential section of a somatic embryo at the heartshaped stage showing no connection of the vascular bundle between the embryo and surface cells of a ray floret Bar: 500 mm

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