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Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch.)
Plant Science 164 (2003) 413 /418 www.elsevier.com/locate/plantsci
Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch.) Young Koung Lee a,b, Won Il Chung b, Hiroshi Ezura a,* a
b
Gene Research Center, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-ku, Taejon 305-701, South Korea Received 13 August 2002; received in revised form 26 November 2002; accepted 27 November 2002
Abstract Using cotyledonary explants excised from seedlings germinated in vitro, efficient plant regeneration via organogenesis was established for two winter squash (Cucurbita maxima Duch.) cultivars. To establish optimal conditions for adventitious shoot induction, a variety of explants were prepared from seedlings of different ages and these were cultured using media containing different concentrations of 6-benzylaminopurine (BA). For both cultivars, plant regeneration was optimal when the proximal parts of cotyledons from 4-day-old seedlings were cultured on induction medium composed of Murashige and Skoog (MS) medium with 1 mg/l BA. After 3 weeks of culture in induction medium, 82 and 92% of explants from the two cultivars regenerated shoots. Adventitious shoots were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA and the elongated shoots were successfully rooted in MS medium without growth regulators for 2 weeks. Flow cytometric analysis revealed that most of the regenerated plants were diploid. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cucurbita maxima Duch; BA; Organogenesis; Flow cytometry
1. Introduction The genus Cucurbita consists of 13 species, including C. pepo L. and C. maxima Duch. C. pepo is important in European and American countries, whereas C. maxima is widely cultivated in East Asian countries, such as China, Japan and Korea. The total area and production of this crop cultivated in China, Japan and Korea in 1999 were 240,000 ha (21%) out of 1,174,000 ha and 3,756,000 tons (25%) out of 15,057,000 tons, respectively (Food and Agriculture Organization of the United Nations, 1999). The development of transformation techniques is important for the exploitation of useful genes for crop improvement and efficient plant regeneration is essential for successful transformation. In the Cucurbitaceae, there have been several reports on the genetic transfor* Corresponding author. Tel./fax: /81-298-537-263. E-mail address: [email protected] (H. Ezura).
mation of Citrullus lanatus [1], Cucumis melo [2], C. pepo [3] and C. sativus [4 /6]. These studies have facilitated target gene transfer into useful crop members of the family. For instance, coat protein-mediated protection of transgenic plants against viruses has been demonstrated via the stable transfer of the coat protein gene of the infectious virus into the plant genome by biolistic transformation of C. melo L. [7]. As another example, three transgenic cucumber strains harboring the rice chitinase gene exhibited enhanced resistance against gray mold [8]. Although physiological, morphological, and cytological studies have been carried out in the winter squash C. maxima Duch. [9 /11], better techniques are needed to facilitate reverse genetic approaches and molecular crop improvement in this species. Previously, cDNAs for the ascorbate peroxidase [12,13], gibberellin 2[b]- and 3[b]hydroxylase [14], copalyl diphosphate synthase [15] and catalase [16] genes have been isolated and characterized in winter squash. In order to elucidate the roles of these genes in winter squash development, a reverse genetic
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(02)00429-6
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approach is required, and for this, efficient plant regeneration and transformation protocols are needed. There have been some reports of regeneration in Cucurbita species, notably in summer squash (C. pepo ). Juretic et al. [17] reported the induction of embryogenic callus derived from summer squash hypocotyl segments and Chee [18] reported that summer squash cultivars were regenerated via somatic embryos produced from shoot apex derived callus. Summer squash cultivars have also been regenerated via somatic embryogenesis using cotyledons [19]. There have been no reports on the successful stable regeneration and transformation of winter squash (C. maxima ), due to the lack of an effective plant regeneration protocol. In this study, we established an efficient regeneration protocol using cotyledonary explants of the winter squash C. maxima . To our knowledge, this is the first report on efficient regeneration via adventitious shoot organogenesis in winter squash.
2. Materials and methods 2.1. Plant materials Two winter squash cultivars, Juktoja (Nongwoo Seed, Suwon, Korea) and Miyako (Sakata Seed, Yokohama, Japan), were used in all experiments. After removal of the seed coat, seeds were sterilized in 1% (v/v) sodium hypochlorite for 20 min, rinsed four times with sterile distilled water and left to air dry in a sterile environment. Seeds were then placed on basal Murashige /
Skoog (MS) [20] medium with 30 g/l sucrose and 3 g/l Gelrite to germinate at 26 8C with a 16-h photoperiod (50 mE m 2 s 1). The pH of all media was adjusted to 5.6 /5.8 prior to the addition of Gelrite and autoclaving at 121 8C for 15 min. Cotyledonary, hypocotyl and root explants were placed on MS medium supplemented with benzylaminopurine (BA) in Petri dishes to induce adventitious shoots. 2.2. Plant regeneration Explants of winter squash seedlings of various ages, ranging from 4 to 9 days post-germination, were cultured on MS medium containing different BA concentrations. Cotyledonary, hypocotyl and root explants of winter squash were excised from in vitro-grown seedlings after germination in darkness. Each cotyledon was cut into eight explants: Hypocotyls and roots were cut into 2 and 1 cm long segments and cultured in Petri dishes containing 50 ml culture medium. Twenty-five explants were placed on each Petri dish containing media with different BA concentrations. For adventitious shoot induction, explants were cultured on MS medium supplemented with 1, 2, 5 or 10 mg/l BA. After 3 weeks of initiation culture, adventitious shoots were transferred onto shoot elongation medium: MS medium containing 0.1 mg/l BA. Elongated shoots were then rooted in half-strength MS medium (1/2 MS). Cultures were kept at 26 8C in a light regime of 16 h light at 50 mE m 2 s 1 and 8 h darkness. After 8 weeks of culture, the number of explants with adventitious shoots was counted.
Table 1 Effect of cotyledon size on adventitious shoot induction from cotyledonary explants of 4-day-old seedlings
Explants cultured on MS medium with 1 mg/l BA were evaluated after 3 weeks of culture. Data are mean values over 11 replications with five petri dish (five explants per petri dish). Values in a column followed by a common letter are significantly different at 5% level by Duncan’s multiple range test.
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2.3. Flow cytometric analysis Nuclei were prepared from the leaves of regenerated plants. Each leaf was chopped with a razor blade in nuclear extraction buffer (Partec, Munster, Germany). The samples were filtered through a 30-mm nylon filter and nuclei in the filtrate were stained with 4,6-diamidino-2-phenylindole (DAPI; Partec). Flow cytometric analysis was performed with a Ploidy Analyzer (Partec).
3. Results 3.1. Plant regeneration Various explants prepared from the cotyledons, hypocotyls and roots of seedlings of two winter squash cultivars were tested for organogenic potential on shoot induction media composed of MS medium supplemented with varying BA concentrations. The shoot induction rate in 4-day-old seedlings of cv. Juktoja was higher than those in 6- and 9-day-old seedlings; and the use of seedlings older than 4 days resulted in a dramatically decreased rate of shoot induction in all explants (Fig. 1A). Among tissues derived from seedlings of the same age, cotyledonary explants containing the most proximal part and with the cotyledon cut in two pieces showed the highest rate of adventitious shoot induction. There was no shoot regeneration in hypocotyls or root explants. The effects of BA concentration at 1, 2, 5 and 10 mg/l were investigated on cotyledonary explants, with 1 /5 mg/l BA producing the highest regeneration. For shoot regeneration, cotyledon age had a more profound effect than BA concentration. Miyako explants showed similar results to Juktoja explants (Fig. 1B).
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Adventitious shoots (Fig. 3A) were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA. The addition of BA to the shoot induction medium increased the efficiency of shoot organogenesis and the production of multiple shoots. A similar result has been described for melon [22]. Overall, the shoot production rate was found to be over 3.2 plantlets per initial cotyledon (data not shown). Elongated shoots (Fig. 3B) were successfully rooted in MS medium without plant growth regulator after 2 weeks (Fig. 3C). Regenerated plantlets were grown to normal plants (Fig. 3D). 3.3. Flow cytometric analysis Flow cytometric analysis of nuclear DNA content was employed to estimate ploidy levels in regenerated plants.
3.2. Effect of cotyledon size and portion In preliminary experiments, shoot induction was affected by the size and the part of the cotyledonary explants used. The effect of cotyledon size and portion on adventitious shoot induction was tested in two cultivars, Juktoja and Miyako (Table 1). Adventitious shoot induction from cotyledons of 4-day-old seedlings cut in two was higher than that from whole, 1/4-, and 1/ 8-sized cotyledons on medium containing 1 mg/l BA. The shoot induction rate was significantly decreased in cotyledon pieces smaller than 1/4-size. This result is similar to the report of Kim et al. [21], in which smaller cotyledons produced fewer adventitious shoots and indicates that cells in the proximal part of the cotyledon are competent for adventitious shoot formation. Of cotyledons excised to half cotyledon size, 82% of Juktoja explants and 92% of Miyako explants individually regenerated shoots after 4 weeks of culture in induction medium (Fig. 2).
Fig. 1. Effects of seedling age and BA concentration on shoot induction from cotyledonary explants in winter squash cvs. Juktoja (A) and Miyako (B). C, Cotyledonary explants; H, hypocotyl explants; R, root explants. Data are mean values over eleven replications with five petri dish (five explants per petri dish). Values in a column followed by a common letter are significantly different at 5% level by Duncan’s multiple range test.
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Fig. 2. Shoot induction and plant regeneration in winter squash. (A) Adventitious shoot induction from the proximal part of cotyledons cultured for 17 days on MS medium with 1 mg/l BA. (B) Shoot elongation on MS medium with 0.1 mg/l BA. (C) Rooted plantlet on 1/2 MS medium without plant growth regulators. (D) Acclimatized plant.
The results of flow cytometric analysis are shown in Table 2. Plantlets regenerated from organogenesis were classified as diploid (2x ), mixoploid (2x/4x) and tetraploid (4x ). In this study, no plantlets of any other ploidy were observed. Among plantlets regenerated using different BA concentrations and cotyledon sizes, diploid (45.8/95.0%), tetraploid (0 /4.2%) and mixoploid (5.0 /50.0%) plants with tetraploid cells were observed. Moreover, cv. Miyako was more affected in ploidy on medium with 1 mg/l BA than cv. Jukyoja, and tetraploid cells were more commonly seen in cv. Miyako than in cv. Juktoja. The size of explants did not greatly affect the ploidy level of regenerated plants, whereas the hormone concentration had a larger effect. The frequency of mixoploids increased with BA concentration in both cultivars. Of 213 adventitious shoots, 73.7% were diploid, 25.8% were mixoploid and 0.5% were tetraploid. Growth of cotyledonary explant regenerants into mature plants varied with ploidy. Diploid regenerants and some of the mixoploid regenerants grew normally into mature plants. The other mixoploid regenerants and one tetraploid regenerant were malformed and did not develop into normal plants.
4. Discussion Using cotyledonary explants excised from seedlings after in vitro germination, efficient plant regeneration via organogenesis was established for two winter squash (C. maxima ) cultivars. The protocol is summarized in Fig. 3. Plant regeneration was optimal when the proximal part of cotyledons from 4-day-old seedlings was cultured on induction medium composed of MS medium with 1 mg/l BA. After 3 weeks of culture in the induction medium, regenerated shoots were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA. Elongated shoots were transferred onto MS medium without plant growth regulators for 2 weeks and rooting plantlets were transplanted into soil. The optimal growth regulator condition for adventitious shoot induction medium was 1 mg/l BA, and a half-cotyledon explant containing the proximal part of the cotyledon showed the highest regeneration efficiency. Regenerated plants were genetically stable in relation to ploidy level. This regeneration system for winter squash shows clear advantages over earlier protocols. First, a higher rate of shoot induction is observed as compared to plant
1a: 1/2 cotyledon cultured on MS medium with 1 mg/l BA; 1: 1/8 cotyledon cultured on MS medium with 1 mg/l BA; 2: 1/8 cotyledon cultured on MS medium with 2 mg/l BA; 5: 1/8 cotyledon cultured on MS medium with 5 mg/l BA; 10: 1/8 cotyledon cultured on MS medium with 10 mg/l BA. Values in a column followed by a common letter are significantly different at 5% level by Duncan’s multiple range test.
1 (4.2)a 0 (0.0)a 0 (0.0)a 0 (0.0)a 0 (0.0)a 12 (50.0)a 9 (39.1)b 7 (38.9)b 7 (28.0)b 4 (16.0)c 11 (45.8)b 14 (60.9)b 11 (61.1)b 21 (84.0)a cv. Miyako No. of plants (%)
18 (72.0)a,b
0 (0.0)a 0 (0.0)a 0 (0.0)a 0 (0.0)a 7 (26.9)a 5 (25.0)a 2 (15.4)a,b 1 (5.3)b 1 (5.0)b 19 (73.1)b 15 (75.0)b 11 (84.6)b 19 (95.0)a cv. Juktoja No. of plants (%)
18 (94.7)a
5 2 1 1a 1a BA (mg/l)
1
2
5
10
1a
1
2
5
10
4x 2x /4x 2x Ploidy
regeneration via somatic embryogenesis. In our study, plant regeneration via organogenesis under optimal conditions occurred in /80% of cotyledonary explants. This frequency is greater than that for somatic embryogenesis in summer squash in the Cucurbita [19]. For example, in the method of Chee [23], only 8% of explants from summer squash produced embryogenic tissue and subsequent plant regeneration efficiency was also low. Another advantage of our method is the lower frequency of polyploid regenerants via our adventitious organogenesis. A high frequency of tetraploidy was observed in melon plants regenerated by adventitious shoot organogenesis [24]. Guis et al. [25] described a highly efficient system for shoot regeneration of melon plants by organogenesis from cotyledonary explants, but /80% of the regenerated plants were found to be tetraploid. In cucumber, Kubalakova et al. [26] found that plants regenerated from callus cultures were a mixture of diploid (57%), tetraploid (18%), octoploid (4%) and mixoploid (2n/4n, 4%; 4n/8n, 17%). In contrast, using our regeneration system based on adventitious shoot induction from cotyledonary explants grown
Table 2 Effect of BA concentration on ploidy levels of plants regenerated from cotyledonary explants of two winter squash cultivars, Juktoja and Miyako
Fig. 3. Steps in plant regeneration via organogenesis from cotyledonary explants of winter squash.
0 (0.0)a
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in vitro, the majority of regenerated winter squash plants were diploid. The mode of regeneration may account for the lower frequency of tetraploid plants. Using our method in winter squash, only one tetraploid plant was obtained from 213 regenerated plants, indicating that this protocol is a highly effective method for the regeneration of diploid plants. Another advantage of our protocol is the speed at which regenerated plants can be produced. Regenerated plants were obtained within 3 months after starting the culture protocol. Adventitious shoots were observed by microscope to regenerate directly from explant tissue, not through callus formation. It is likely that this direct regeneration results in the rapid plant regeneration. If somatic embryogenesis is required as a mode of plant regeneration, it usually takes more than 6 months to produce whole plantlets in C. pepo [19]. In conclusion, we believe that this stable and rapid shoot regeneration system for diploid winter squash will contribute to the efficient production of transgenic plants, and that the method will be used not only for fundamental studies of winter squash genes, but also as a practical tool for winter squash improvement.
[8]
[9] [10]
[11] [12]
[13]
[14]
[15]
[16]
Acknowledgements This work was supported by the Brain Korea 21 Project in 2001 and, in part, by a grant-in-aid from the ‘Research for the Future’ program of the Japan Society for the Promotion of Science (JSPS-RFTF97L00601).
[17]
[18]
[19]
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Efficient plant regeneration via organogenesis in winter squash (Cucurbita maxima Duch.) Young Koung Lee a,b, Won Il Chung b, Hiroshi Ezura a,* a
b
Gene Research Center, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusong-dong, Yusong-ku, Taejon 305-701, South Korea Received 13 August 2002; received in revised form 26 November 2002; accepted 27 November 2002
Abstract Using cotyledonary explants excised from seedlings germinated in vitro, efficient plant regeneration via organogenesis was established for two winter squash (Cucurbita maxima Duch.) cultivars. To establish optimal conditions for adventitious shoot induction, a variety of explants were prepared from seedlings of different ages and these were cultured using media containing different concentrations of 6-benzylaminopurine (BA). For both cultivars, plant regeneration was optimal when the proximal parts of cotyledons from 4-day-old seedlings were cultured on induction medium composed of Murashige and Skoog (MS) medium with 1 mg/l BA. After 3 weeks of culture in induction medium, 82 and 92% of explants from the two cultivars regenerated shoots. Adventitious shoots were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA and the elongated shoots were successfully rooted in MS medium without growth regulators for 2 weeks. Flow cytometric analysis revealed that most of the regenerated plants were diploid. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cucurbita maxima Duch; BA; Organogenesis; Flow cytometry
1. Introduction The genus Cucurbita consists of 13 species, including C. pepo L. and C. maxima Duch. C. pepo is important in European and American countries, whereas C. maxima is widely cultivated in East Asian countries, such as China, Japan and Korea. The total area and production of this crop cultivated in China, Japan and Korea in 1999 were 240,000 ha (21%) out of 1,174,000 ha and 3,756,000 tons (25%) out of 15,057,000 tons, respectively (Food and Agriculture Organization of the United Nations, 1999). The development of transformation techniques is important for the exploitation of useful genes for crop improvement and efficient plant regeneration is essential for successful transformation. In the Cucurbitaceae, there have been several reports on the genetic transfor* Corresponding author. Tel./fax: /81-298-537-263. E-mail address: [email protected] (H. Ezura).
mation of Citrullus lanatus [1], Cucumis melo [2], C. pepo [3] and C. sativus [4 /6]. These studies have facilitated target gene transfer into useful crop members of the family. For instance, coat protein-mediated protection of transgenic plants against viruses has been demonstrated via the stable transfer of the coat protein gene of the infectious virus into the plant genome by biolistic transformation of C. melo L. [7]. As another example, three transgenic cucumber strains harboring the rice chitinase gene exhibited enhanced resistance against gray mold [8]. Although physiological, morphological, and cytological studies have been carried out in the winter squash C. maxima Duch. [9 /11], better techniques are needed to facilitate reverse genetic approaches and molecular crop improvement in this species. Previously, cDNAs for the ascorbate peroxidase [12,13], gibberellin 2[b]- and 3[b]hydroxylase [14], copalyl diphosphate synthase [15] and catalase [16] genes have been isolated and characterized in winter squash. In order to elucidate the roles of these genes in winter squash development, a reverse genetic
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(02)00429-6
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Y.K. Lee et al. / Plant Science 164 (2003) 413 /418
approach is required, and for this, efficient plant regeneration and transformation protocols are needed. There have been some reports of regeneration in Cucurbita species, notably in summer squash (C. pepo ). Juretic et al. [17] reported the induction of embryogenic callus derived from summer squash hypocotyl segments and Chee [18] reported that summer squash cultivars were regenerated via somatic embryos produced from shoot apex derived callus. Summer squash cultivars have also been regenerated via somatic embryogenesis using cotyledons [19]. There have been no reports on the successful stable regeneration and transformation of winter squash (C. maxima ), due to the lack of an effective plant regeneration protocol. In this study, we established an efficient regeneration protocol using cotyledonary explants of the winter squash C. maxima . To our knowledge, this is the first report on efficient regeneration via adventitious shoot organogenesis in winter squash.
2. Materials and methods 2.1. Plant materials Two winter squash cultivars, Juktoja (Nongwoo Seed, Suwon, Korea) and Miyako (Sakata Seed, Yokohama, Japan), were used in all experiments. After removal of the seed coat, seeds were sterilized in 1% (v/v) sodium hypochlorite for 20 min, rinsed four times with sterile distilled water and left to air dry in a sterile environment. Seeds were then placed on basal Murashige /
Skoog (MS) [20] medium with 30 g/l sucrose and 3 g/l Gelrite to germinate at 26 8C with a 16-h photoperiod (50 mE m 2 s 1). The pH of all media was adjusted to 5.6 /5.8 prior to the addition of Gelrite and autoclaving at 121 8C for 15 min. Cotyledonary, hypocotyl and root explants were placed on MS medium supplemented with benzylaminopurine (BA) in Petri dishes to induce adventitious shoots. 2.2. Plant regeneration Explants of winter squash seedlings of various ages, ranging from 4 to 9 days post-germination, were cultured on MS medium containing different BA concentrations. Cotyledonary, hypocotyl and root explants of winter squash were excised from in vitro-grown seedlings after germination in darkness. Each cotyledon was cut into eight explants: Hypocotyls and roots were cut into 2 and 1 cm long segments and cultured in Petri dishes containing 50 ml culture medium. Twenty-five explants were placed on each Petri dish containing media with different BA concentrations. For adventitious shoot induction, explants were cultured on MS medium supplemented with 1, 2, 5 or 10 mg/l BA. After 3 weeks of initiation culture, adventitious shoots were transferred onto shoot elongation medium: MS medium containing 0.1 mg/l BA. Elongated shoots were then rooted in half-strength MS medium (1/2 MS). Cultures were kept at 26 8C in a light regime of 16 h light at 50 mE m 2 s 1 and 8 h darkness. After 8 weeks of culture, the number of explants with adventitious shoots was counted.
Table 1 Effect of cotyledon size on adventitious shoot induction from cotyledonary explants of 4-day-old seedlings
Explants cultured on MS medium with 1 mg/l BA were evaluated after 3 weeks of culture. Data are mean values over 11 replications with five petri dish (five explants per petri dish). Values in a column followed by a common letter are significantly different at 5% level by Duncan’s multiple range test.
Y.K. Lee et al. / Plant Science 164 (2003) 413 /418
2.3. Flow cytometric analysis Nuclei were prepared from the leaves of regenerated plants. Each leaf was chopped with a razor blade in nuclear extraction buffer (Partec, Munster, Germany). The samples were filtered through a 30-mm nylon filter and nuclei in the filtrate were stained with 4,6-diamidino-2-phenylindole (DAPI; Partec). Flow cytometric analysis was performed with a Ploidy Analyzer (Partec).
3. Results 3.1. Plant regeneration Various explants prepared from the cotyledons, hypocotyls and roots of seedlings of two winter squash cultivars were tested for organogenic potential on shoot induction media composed of MS medium supplemented with varying BA concentrations. The shoot induction rate in 4-day-old seedlings of cv. Juktoja was higher than those in 6- and 9-day-old seedlings; and the use of seedlings older than 4 days resulted in a dramatically decreased rate of shoot induction in all explants (Fig. 1A). Among tissues derived from seedlings of the same age, cotyledonary explants containing the most proximal part and with the cotyledon cut in two pieces showed the highest rate of adventitious shoot induction. There was no shoot regeneration in hypocotyls or root explants. The effects of BA concentration at 1, 2, 5 and 10 mg/l were investigated on cotyledonary explants, with 1 /5 mg/l BA producing the highest regeneration. For shoot regeneration, cotyledon age had a more profound effect than BA concentration. Miyako explants showed similar results to Juktoja explants (Fig. 1B).
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Adventitious shoots (Fig. 3A) were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA. The addition of BA to the shoot induction medium increased the efficiency of shoot organogenesis and the production of multiple shoots. A similar result has been described for melon [22]. Overall, the shoot production rate was found to be over 3.2 plantlets per initial cotyledon (data not shown). Elongated shoots (Fig. 3B) were successfully rooted in MS medium without plant growth regulator after 2 weeks (Fig. 3C). Regenerated plantlets were grown to normal plants (Fig. 3D). 3.3. Flow cytometric analysis Flow cytometric analysis of nuclear DNA content was employed to estimate ploidy levels in regenerated plants.
3.2. Effect of cotyledon size and portion In preliminary experiments, shoot induction was affected by the size and the part of the cotyledonary explants used. The effect of cotyledon size and portion on adventitious shoot induction was tested in two cultivars, Juktoja and Miyako (Table 1). Adventitious shoot induction from cotyledons of 4-day-old seedlings cut in two was higher than that from whole, 1/4-, and 1/ 8-sized cotyledons on medium containing 1 mg/l BA. The shoot induction rate was significantly decreased in cotyledon pieces smaller than 1/4-size. This result is similar to the report of Kim et al. [21], in which smaller cotyledons produced fewer adventitious shoots and indicates that cells in the proximal part of the cotyledon are competent for adventitious shoot formation. Of cotyledons excised to half cotyledon size, 82% of Juktoja explants and 92% of Miyako explants individually regenerated shoots after 4 weeks of culture in induction medium (Fig. 2).
Fig. 1. Effects of seedling age and BA concentration on shoot induction from cotyledonary explants in winter squash cvs. Juktoja (A) and Miyako (B). C, Cotyledonary explants; H, hypocotyl explants; R, root explants. Data are mean values over eleven replications with five petri dish (five explants per petri dish). Values in a column followed by a common letter are significantly different at 5% level by Duncan’s multiple range test.
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Fig. 2. Shoot induction and plant regeneration in winter squash. (A) Adventitious shoot induction from the proximal part of cotyledons cultured for 17 days on MS medium with 1 mg/l BA. (B) Shoot elongation on MS medium with 0.1 mg/l BA. (C) Rooted plantlet on 1/2 MS medium without plant growth regulators. (D) Acclimatized plant.
The results of flow cytometric analysis are shown in Table 2. Plantlets regenerated from organogenesis were classified as diploid (2x ), mixoploid (2x/4x) and tetraploid (4x ). In this study, no plantlets of any other ploidy were observed. Among plantlets regenerated using different BA concentrations and cotyledon sizes, diploid (45.8/95.0%), tetraploid (0 /4.2%) and mixoploid (5.0 /50.0%) plants with tetraploid cells were observed. Moreover, cv. Miyako was more affected in ploidy on medium with 1 mg/l BA than cv. Jukyoja, and tetraploid cells were more commonly seen in cv. Miyako than in cv. Juktoja. The size of explants did not greatly affect the ploidy level of regenerated plants, whereas the hormone concentration had a larger effect. The frequency of mixoploids increased with BA concentration in both cultivars. Of 213 adventitious shoots, 73.7% were diploid, 25.8% were mixoploid and 0.5% were tetraploid. Growth of cotyledonary explant regenerants into mature plants varied with ploidy. Diploid regenerants and some of the mixoploid regenerants grew normally into mature plants. The other mixoploid regenerants and one tetraploid regenerant were malformed and did not develop into normal plants.
4. Discussion Using cotyledonary explants excised from seedlings after in vitro germination, efficient plant regeneration via organogenesis was established for two winter squash (C. maxima ) cultivars. The protocol is summarized in Fig. 3. Plant regeneration was optimal when the proximal part of cotyledons from 4-day-old seedlings was cultured on induction medium composed of MS medium with 1 mg/l BA. After 3 weeks of culture in the induction medium, regenerated shoots were subcultured on elongation medium composed of MS medium with 0.1 mg/l BA. Elongated shoots were transferred onto MS medium without plant growth regulators for 2 weeks and rooting plantlets were transplanted into soil. The optimal growth regulator condition for adventitious shoot induction medium was 1 mg/l BA, and a half-cotyledon explant containing the proximal part of the cotyledon showed the highest regeneration efficiency. Regenerated plants were genetically stable in relation to ploidy level. This regeneration system for winter squash shows clear advantages over earlier protocols. First, a higher rate of shoot induction is observed as compared to plant
1a: 1/2 cotyledon cultured on MS medium with 1 mg/l BA; 1: 1/8 cotyledon cultured on MS medium with 1 mg/l BA; 2: 1/8 cotyledon cultured on MS medium with 2 mg/l BA; 5: 1/8 cotyledon cultured on MS medium with 5 mg/l BA; 10: 1/8 cotyledon cultured on MS medium with 10 mg/l BA. Values in a column followed by a common letter are significantly different at 5% level by Duncan’s multiple range test.
1 (4.2)a 0 (0.0)a 0 (0.0)a 0 (0.0)a 0 (0.0)a 12 (50.0)a 9 (39.1)b 7 (38.9)b 7 (28.0)b 4 (16.0)c 11 (45.8)b 14 (60.9)b 11 (61.1)b 21 (84.0)a cv. Miyako No. of plants (%)
18 (72.0)a,b
0 (0.0)a 0 (0.0)a 0 (0.0)a 0 (0.0)a 7 (26.9)a 5 (25.0)a 2 (15.4)a,b 1 (5.3)b 1 (5.0)b 19 (73.1)b 15 (75.0)b 11 (84.6)b 19 (95.0)a cv. Juktoja No. of plants (%)
18 (94.7)a
5 2 1 1a 1a BA (mg/l)
1
2
5
10
1a
1
2
5
10
4x 2x /4x 2x Ploidy
regeneration via somatic embryogenesis. In our study, plant regeneration via organogenesis under optimal conditions occurred in /80% of cotyledonary explants. This frequency is greater than that for somatic embryogenesis in summer squash in the Cucurbita [19]. For example, in the method of Chee [23], only 8% of explants from summer squash produced embryogenic tissue and subsequent plant regeneration efficiency was also low. Another advantage of our method is the lower frequency of polyploid regenerants via our adventitious organogenesis. A high frequency of tetraploidy was observed in melon plants regenerated by adventitious shoot organogenesis [24]. Guis et al. [25] described a highly efficient system for shoot regeneration of melon plants by organogenesis from cotyledonary explants, but /80% of the regenerated plants were found to be tetraploid. In cucumber, Kubalakova et al. [26] found that plants regenerated from callus cultures were a mixture of diploid (57%), tetraploid (18%), octoploid (4%) and mixoploid (2n/4n, 4%; 4n/8n, 17%). In contrast, using our regeneration system based on adventitious shoot induction from cotyledonary explants grown
Table 2 Effect of BA concentration on ploidy levels of plants regenerated from cotyledonary explants of two winter squash cultivars, Juktoja and Miyako
Fig. 3. Steps in plant regeneration via organogenesis from cotyledonary explants of winter squash.
0 (0.0)a
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in vitro, the majority of regenerated winter squash plants were diploid. The mode of regeneration may account for the lower frequency of tetraploid plants. Using our method in winter squash, only one tetraploid plant was obtained from 213 regenerated plants, indicating that this protocol is a highly effective method for the regeneration of diploid plants. Another advantage of our protocol is the speed at which regenerated plants can be produced. Regenerated plants were obtained within 3 months after starting the culture protocol. Adventitious shoots were observed by microscope to regenerate directly from explant tissue, not through callus formation. It is likely that this direct regeneration results in the rapid plant regeneration. If somatic embryogenesis is required as a mode of plant regeneration, it usually takes more than 6 months to produce whole plantlets in C. pepo [19]. In conclusion, we believe that this stable and rapid shoot regeneration system for diploid winter squash will contribute to the efficient production of transgenic plants, and that the method will be used not only for fundamental studies of winter squash genes, but also as a practical tool for winter squash improvement.
[8]
[9] [10]
[11] [12]
[13]
[14]
[15]
[16]
Acknowledgements This work was supported by the Brain Korea 21 Project in 2001 and, in part, by a grant-in-aid from the ‘Research for the Future’ program of the Japan Society for the Promotion of Science (JSPS-RFTF97L00601).
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