- Email: [email protected]
Characterization of two-step direct somatic embryogenesis in carrot
Biochemical Engineering Journal 38 (2008) 206–211
Characterization of two-step direct somatic embryogenesis in carrot Toshiya Takeda a,b,∗ , Miki Mizukami b , Hiroshi Matsuoka b a
Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, 1-50-1 Mutsuura-Higashi, Kanazawa-ku, Yokohama 236-8501, Japan b Department of Biosciences, Teikyo University of Science and Technology, Uenohara, Yamanashi 409-0193, Japan Received 28 December 2006; received in revised form 31 May 2007; accepted 3 July 2007
Abstract Direct somatic embryogenesis has been used to induce intact regenerated plantlets. We applied two-step direct somatic embryogenesis to the mass propagation of carrots and characterized this method. Morphological observation revealed that somatic embryos were generated from whole segments except for the callus part. Secondarily formed collenchyma-like structures were observed in the segments after the induction culture. The somatic embryos were generated from the collenchyma-like structure. When the longer-grown hypocotyls which had less potential to generate somatic embryos were used, the collenchyma-like structure did not develop. RAPD-PCR investigation showed that the regenerated plantlets had genetic stability. These results indicated that two-step direct somatic embryogenesis was a useful method for the mass propagation of plants without losing the quality of the donor plants. © 2007 Elsevier B.V. All rights reserved. Keywords: Direct somatic embryo; Carrot; Plant cell culture; Tissue cell culture; Genetic stability; DNA
1. Introduction Direct somatic embryogenesis (DSE), which induces somatic embryos directly from donor plantlets, was supposed to be associated with the cytological and genetic stability of regenerated plantlets. Some research with random amplified polymorphic DNA (RAPD) has revealed genetic stability in DSE [1–3]. The DSE procedures can be categorized into two groups: one-step procedures and two-step procedures. In one-step DSE, somatic embryos are formed with a single culture process using donor tissue which has high regeneration potential, for example zygotic cotyledon [4–6]. On the other hand, two-step DSE, which consists of induction and development cultures, can be initiated from hypocotyl or leaf segments. We investigated the high productivity of regenerated plantlets in the two-step DSE system in our previous paper [7]. We succeeded in inducing 1700 carrot plantlets from a hypocotyl. This number was much larger than the reported values for one-step DSE in various plant species.
∗ Corresponding author at: Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, 1-50-1 Mutsuura-Higashi, Kanazawa-ku, Yokohama 236-8501, Japan. Tel.: +81 45 784 8153. E-mail address: [email protected] (T. Takeda).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.07.006
The induction culture in two-step DSE includes an auxin treatment similar to somatic embryogenesis via callus cells. The difference between the procedures of the two-step DSE and callus-mediated somatic embryogenesis is merely the cultivation period with auxins. Tokuji et al. also reported DSE in the carrot [8–10]. They were able to induce the direct somatic embryo with a short time exposure to a high concentration of 2,4-dichlorophenoxy acetic acid (2,4-D) within 24–48 h [9]. In this treatment, the donor plantlet kept its initial shape, and the somatic embryos were generated directly from epidermal cells. On the other hand, in our study induction culture was carried out for 2 weeks. This induction culture expanded the donor plantlet and increased the number of regenerated plantlets. If the induction culture were prolonged, somatic embryos might be generated from the callus rather than from donor plant tissues. Also, long exposure of the plant tissues to 2,4-D was reported to cause somatic variations [11–13]. Therefore, prior to applying the two-step DSE, it should be confirmed whether the somatic embryos were generated from origins other than callus cells and whether somatic variation was suppressed during the induction culture. The present article is concerned with the quality of this system as a propagation system. We carried out histological observation and analysis of RAPD in two-step DSE using carrot hypocotyls.
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2. Materials and methods
2.2. Histological observation
2.1. Cells and medium
Morphological development in the course of the cultures was observed under a stereo microscope equipped with a digital camera on the torinocular port. Thin-sectioned segment samples were observed under a differential interference microscope for histological observation. Samples used for thin sectioning were fixed with 7.2% formaldehyde in 50 mM sodium phosphate buffer with 0.1% IGEPAL CA-630 (MP Biomedicals, Inc.) and 10% dimethyl sulfoxide for 40 min at room temperature. They were embedded in O.C.T. (Optical Cutting Temperature) compound (Tissue Tek) and frozen in liquid nitrogen. Frozen sections of 5-m thickness were prepared using a cryostat (Leica CM1850, Nussloch, Germany). The sections were placed on slide glasses shredded with 3% low-melting temperature agarose. The segments were fixed with a mixture of 38% formaldehyde solution, acetic acid and 70% ethanol (1:1:18), and freeze sec-
Seeds of carrot (Daucus carota L. cv. Natsumaki-Senkougosun) were surface-sterilized by soaking in 70% (v/v) ethanol for 3 min and then in 1% active sodium hypochlorite solution for 30 min. They were washed extensively in sterile water and then allowed to germinate for 1–3 weeks at 26 ◦ C in darkness on Murashige and Skoog (MS) solid medium with 8 g L−1 of agar without sucrose. Hypocotyl explants without apical meristems were removed from the seedlings and cut into 0.5 cm segments. The hypocotyl segments were placed on MS solid medium with 0.5 mg L−1 of 2,4-D, 30 g L−1 of sucrose and 8 g L−1 of agar. They were incubated at 26 ◦ C under continuous light of 2000 Lx (induction culture). After 14 days, the segments were transferred to new MS solid medium without 2,4-D and incubated for another 30 days (development culture).
Fig. 1. Typical morphological change in a hypocotyl segment during two-step DSE. A segment was incubated for 44 days, which included the first 14 days of the induction culture and the following 30 days of the development culture. The culture periods of the shown segment were 0 days (a), 7 days (b), 14 days (c), 21 days (d), 25 days (e) and 28 days (f). The numbers in (d) represent the indices for estimating the dependency of somatic embryogenesis on the location in a segment, as shown in Fig. 2.
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tions were prepared for propidium iodide (PI) staining. Twenty microliter of PI working solution, which contained 5 mg L−1 of PI in PBS buffer, was dropped on the frozen section [14]. The section was incubated at room temperature for 10 min, rinsed with PBS buffer and observed under a confocal laser microscope (Olympus FV300, Tokyo, Japan). 2.3. Estimation of genetic stability by RAPD-PCR The genetic stability of the regenerated plants was estimated using RAPD-PCR. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen K.K.) according to the manufacturer’s protocol from hypocotyl segments after cutting, from segments after 2 weeks of induction culture, from regenerated plantlets after a further 4 weeks of developmental culture, and from callus-forming segments after 6 weeks of induction culture. A set of these samples was prepared from one seedling. Their RAPD patterns were compared using four sets of samples. RAPD-PCR was carried out using the Ready-To-Go RAPD Analysis Kit (Amersham Pharmacia Biotech) according to the
Fig. 2. Dependency of somatic embryogenesis on the location in a donor segment. The numbers of regenerated plantlets were counted in each location of a segment that was fractioned and numbered from the root side as shown in Fig. 1(d). The ratios of regenerated plantlets in each location to the total count were evaluated based on the observation of four hypocotyl segments.
Fig. 3. Cross-sections of hypocotyls before cultivation (a and b) and after 14 days of induction culture (c and d). The donor hypocotyls were 3 cm (a and c) and 6 cm (b and d) in length. Abbreviations: v, vascular tissue; co, cortex; ep, epidermis; cav, cavity.
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manufacturer’s protocol. Five primers including the kit (No. 1–5) were used. The condition for the PCR was 95 ◦ C for 5 min (initial melting) and 45 cycles of 95 ◦ C for 60 s, 36 ◦ C for 60 s, and 72 ◦ C for 120 s. The PCR products were electrophoresed on 3.0% agarose gels (Nusieve 3:1 Agarose) and were stained with ethidium bromide. 3. Results and discussion 3.1. Morphological observation of somatic embryogenesis Fig. 1 shows a series of typical morphological changes in a hypocotyl segment during two-step DSE. The segment expanded during the first week (a and b). After 14 days (c), the epidermal tissue became transparent and the vascular tissue was yellowed. The edges of the segment expanded and yellowed. The segment was transferred to the development medium on the 14th day. After 25 days (11 days in the development culture) somatic embryos were generated from under the transparent epidermal cells (e). Plantlets from the somatic embryos developed and covered the donor segments in a 44-day culture (30-day development culture). The plantlets were 1–3 cm in length and could be used as donors for the next direct somatic embryogenesis. The dependency of somatic embryogenesis on the location in a donor segment was illustrated in Fig. 2. The number of plantlets appearing on photograph was counted after 11 days of development culture. The average total number of plantlets from a segment was 24. At the end of the culture, 4–5 times more plantlets were observed than at the 11th day of the development culture. However, the origin of the plantlets could not be determined at the end of the cultures. Callus formed on each edge of the segment and somatic embryos were generated from the whole segment except for the callus part. A high frequency of regenerated somatic embryos was observed near one edge of the segments. The edge did not, however, depend on the polarity of the donor plants. Therefore the deviation for the number of somatic embryos generated from the ends of the segments was bigger than those for other data. Shortening the donor segments to 0.2 cm did not influence the frequency of somatic embryogenesis (data not shown). 3.2. Histological observation
Fig. 4. Thin section of hypocotyl segment in the development culture with a thick cell wall structure. The structure was observed at the outer part next to the epidermis on the 5th day of the development culture (a). This structure was observed to be fibrous in axial section. Somatic embryos were observed on the structure at the 7th day of development culture (b). Abbreviations: v, vascular tissue; co, cortex; ep, epidermis; tw, structure with a thick cell wall; se, somatic embryo.
Histological observation was carried out to determine the location from which somatic embryos were generated. Two groups of donor hypocotyls with different lengths were compared. We found that the number of somatic embryos generated from a segment decreased as the hypocotyl length of the seedling increased [7]. Fig. 3(a–d) shows cross-sections of two of the donor hypocotyls before cultivation and after 14 days of induction culture. These hypocotyls were 3 and 6 cm in length. Cortex with parenchyma cells filled between the epidermis and vascular tissue. The number of parenchyma cells was smaller in the longer hypocotyl than the shorter one before the culture (Figs. 3a, 3b).
After 14 days, a cavity was observed in the longer hypocotyl (Fig. 3d). A structure with a thick cell wall was formed at the outer part next to the epidermis on the 5th day in the development culture as shown in Fig. 4a. This structure was observed to be fibrous in the axial section. The formations of this structure were observed in all segments from the 3-cm hypocotyls, although the time to form the structure varied with the individual hypocotyl from the 10th day of the induction culture to the 5th day of the development culture. And it could not be observed in segments from 6-cm hypocotyls. Somatic embryos were observed on the
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Fig. 5. Cross-section of a segment stained with propidium iodide after 10 days of development culture. Fluorescence was observed in the somatic embryos and in the structure with a thick cell wall. Abbreviations: v, vascular tissue; ep, epidermis; tw, structure with a thick cell wall; se, somatic embryo.
fibrous structure on the 7th day of the development culture as shown in Fig. 4b. Fig. 5 showed a cross-section of a segment after 10 days of development culture staining with PI. The fluorescence of PI, which stains nucleotides, was observed in the somatic embryos and in the structure with a thick cell wall. From these observations, this structure was concluded to be similar to collenchyma, because it was fibrous, surrounded by a thick cell wall, and contained rich nucleotides. This collenchyma-like structure is necessary to generate somatic embryos, judging from the results showing that the somatic embryos were generated from the structure and the structure was not observed in 6-cm-long hypocotyls which had a low potential to generate somatic embryos. The epidermal cells may differentiate and prolong along with the development of the hypocotyls. The growth of the parenchyma cells may not be able to catch up with the increase in the length. Thus, the density of the parenchyma cells may decrease in the longer hypocotyls. We think that the parenchyma cells with low density would part from the epidermis during the induction culture and that a cavity would be generated. The
Fig. 6. A representative banding pattern of RAPD-PCR products using template DNAs from donor hypocotyls (D), induction-cultured segments (I), regenerated plantlets (R) and callus-forming segments (C). Two PCR products (arrow) disappeared with the callus-forming segments.
parenchyma cells that are separated from the epidermis might not be able to form the fibrous structure. Masuda et al. reported that somatic embryos were generated directly from the epidermal cells of donor explants when the shorter treatment with 2,4-D was used [8]. In our two-step DSE, somatic embryos were generated from the secondary formed collenchyma-like structure. This observation locates the twostep DSE between the direct somatic embryogenesis and the callus-mediated somatic embryogenesis. 3.3. Analysis of somaclonal variation The genetic stability was evaluated with RAPD-PCR. The PCR was carried out using the four sets of hypocotyls with five primers. The number of amplified DNA fragments, which ranged in size from 300 to 1500 bp, ranged from 4 to 12 depending on the primer that was used. Fig. 6 shows a rep-
Table 1 Observed number of PCR products
Total number of PCR products from donor hypocotyls Number of disappeared bandsa Number of emerged bandsa Mutation index (%)b a b
Induction cultured segments
Regenerated plantlets
0 0 0.0
1 0 0.6
Callus forming segments
169
The number of disappeared and emerged bands compared to the donor hypocotyls. Mutation index was the ratio of disappeared and emerged bands to the observed PCR products with donor hypocotyls.
21 0 12.4
T. Takeda et al. / Biochemical Engineering Journal 38 (2008) 206–211
resentative banding pattern of PCR products amplified using template DNAs from a donor hypocotyl, an induction-cultured segment, a regenerated plantlet and a callus-forming segment. Table 1 summarizes the observed number of PCR products and the number of disappeared and emerged bands in comparison to the donor hypocotyls. A mutation index was defined as the ratio of disappeared and emerged bands to the observed PCR products with the donor hypocotyls. The PCR products counted 169 with the donor hypocotyls. Only 1 product disappeared with the regenerated plantlets, and 21 products disappeared with the callus-forming segments. No change was observed with the induction-cultured segments. No emerged products were observed in all cases. The mutation indexes were 0, 0.6 and 12% for the inductioncultured segments, regenerated plantlets, and callus-forming segments, respectively. Therefore, the plantlets induced with two-step DSE will have higher genetic stability than callus cells. 4. Conclusion We characterized two-step DSE using carrot hypocotyls. RAPD-PCR research revealed that this method has genetic stability similar to other direct somatic embryogenesis systems. This genetic stability of the regenerated plantlets was supposed to be related to the origin of the somatic embryos. The somatic embryos were generated from the secondary structure, a collenchyma-like structure, rather than from callus cells as observed in this study. Furthermore, somatic embryogenesis from the secondary structure may have caused the frequency of regeneration to be greater than that of one-step direct somatic embryogenesis and other organogenesis systems. Acknowledgments This research was supported in part by a Grant-in-Aid for Scientific Research (No. 12750707) and a Grant-in-Aid for Advanced Scientific Research on Bioscience/Biotechnology Areas from the Ministry of Education, Science, Sports and Culture of Japan.
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References [1] Y. Shoyama, X.X. Zhu, R. Nakai, S. Shiraishi, H. Kohda, Micropropagation of Panax notoginseng by somatic embryogenesis and RAPD analysis of regeneration plantlets, Plant Cell Rep. 16 (1997) 450–453. [2] J.L. Fourre, P. Berger, L. Niquet, P. Andre, Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches, Ther. Appl. Genet. 94 (1997) 159–169. [3] P.C. Binsfeld, R. Wingender, J. Wunder, H. Schnabl, Direct embryogenesis in the genus Helianthus and RAPD analysis of obtained clones, J. Appl. Bot.-Angewandte Botanik 73 (1999) 63–68. [4] A. Nadolska-Orczyk, W. Orczyc, New aspects of soybean somatic embryogenesis, Euphytica 80 (1994) 137–143. [5] S. Kulshreshtha, R.H.A. Coutts, Direct somatic embryogenesis and plant regeneration from mature sugarbeet (Beta vulgaris L.) zygotic cotyledons, Plant Growth Regul. 22 (1997) 87–92. [6] Y.E. Choi, D.C. Yang, J.C. Park, W.Y. Soh, K.T. Choi, Regenerative ability of somatic single and multiple embryos from cotyledons of Korean ginseng on hormone-free medium, Plant Cell Reports 17 (1998) 544– 551. [7] M. Mizukami, T. Takeda, H. Satonaka, H. Matsuoka, Improvement of propagation frequency with two-step direct somatic embryogenesis, Biochem. Eng. J. 38 (2008) 55–60. [8] H. Masuda, S. Oohashi, Y. Tokuji, Y. Mizue, Direct embryo formation from epidermal cells of carrot hypocotyls, J. Plant Physiol. 145 (1995) 531– 534. [9] Y. Tokuji, H. Masuda, Duration of treatment of carrot hypocotyl explants with 2,4-dichlorophenoxyacetic acid for direct somatic embryogenesis, Biosci. Biotechnol. Biochem. 60 (1996) 891–892. [10] Y. Tokuji, H. Fukuda, A rapid method for transformation of carrot (Daucus carota L.) by using direct somatic embryogenesis, Biosci. Biotechnol. Biochem. 63 (1999) 519–523. [11] P. Lakin, W. Scowcroft, Somaclonal variation: a novel source of variability from cell cultures for plant improvement, Theor. Appl. Genet. 60 (1981) 197–214. [12] M. Pavlica, D. Papes, B. Nagy, 2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells, Mutant. Res. 263 (1991) 77–81. [13] R.S. Sangwan, Y. Bourgeois, B.S. Sangwan-Norreel, Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency, Mol. Gen. Genet. 230 (1991) 475–485. [14] S. Matsunaga, M. Hizume, S. Kawano, T. Kuroiwa, Cytological analyses of Melandrium album: genome size, chromosome size and fluorescence in situ hybridization, Cytologia 59 (1994) 135–141.
Characterization of two-step direct somatic embryogenesis in carrot Toshiya Takeda a,b,∗ , Miki Mizukami b , Hiroshi Matsuoka b a
Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, 1-50-1 Mutsuura-Higashi, Kanazawa-ku, Yokohama 236-8501, Japan b Department of Biosciences, Teikyo University of Science and Technology, Uenohara, Yamanashi 409-0193, Japan Received 28 December 2006; received in revised form 31 May 2007; accepted 3 July 2007
Abstract Direct somatic embryogenesis has been used to induce intact regenerated plantlets. We applied two-step direct somatic embryogenesis to the mass propagation of carrots and characterized this method. Morphological observation revealed that somatic embryos were generated from whole segments except for the callus part. Secondarily formed collenchyma-like structures were observed in the segments after the induction culture. The somatic embryos were generated from the collenchyma-like structure. When the longer-grown hypocotyls which had less potential to generate somatic embryos were used, the collenchyma-like structure did not develop. RAPD-PCR investigation showed that the regenerated plantlets had genetic stability. These results indicated that two-step direct somatic embryogenesis was a useful method for the mass propagation of plants without losing the quality of the donor plants. © 2007 Elsevier B.V. All rights reserved. Keywords: Direct somatic embryo; Carrot; Plant cell culture; Tissue cell culture; Genetic stability; DNA
1. Introduction Direct somatic embryogenesis (DSE), which induces somatic embryos directly from donor plantlets, was supposed to be associated with the cytological and genetic stability of regenerated plantlets. Some research with random amplified polymorphic DNA (RAPD) has revealed genetic stability in DSE [1–3]. The DSE procedures can be categorized into two groups: one-step procedures and two-step procedures. In one-step DSE, somatic embryos are formed with a single culture process using donor tissue which has high regeneration potential, for example zygotic cotyledon [4–6]. On the other hand, two-step DSE, which consists of induction and development cultures, can be initiated from hypocotyl or leaf segments. We investigated the high productivity of regenerated plantlets in the two-step DSE system in our previous paper [7]. We succeeded in inducing 1700 carrot plantlets from a hypocotyl. This number was much larger than the reported values for one-step DSE in various plant species.
∗ Corresponding author at: Department of Applied Material and Life Science, College of Engineering, Kanto Gakuin University, 1-50-1 Mutsuura-Higashi, Kanazawa-ku, Yokohama 236-8501, Japan. Tel.: +81 45 784 8153. E-mail address: [email protected] (T. Takeda).
1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.07.006
The induction culture in two-step DSE includes an auxin treatment similar to somatic embryogenesis via callus cells. The difference between the procedures of the two-step DSE and callus-mediated somatic embryogenesis is merely the cultivation period with auxins. Tokuji et al. also reported DSE in the carrot [8–10]. They were able to induce the direct somatic embryo with a short time exposure to a high concentration of 2,4-dichlorophenoxy acetic acid (2,4-D) within 24–48 h [9]. In this treatment, the donor plantlet kept its initial shape, and the somatic embryos were generated directly from epidermal cells. On the other hand, in our study induction culture was carried out for 2 weeks. This induction culture expanded the donor plantlet and increased the number of regenerated plantlets. If the induction culture were prolonged, somatic embryos might be generated from the callus rather than from donor plant tissues. Also, long exposure of the plant tissues to 2,4-D was reported to cause somatic variations [11–13]. Therefore, prior to applying the two-step DSE, it should be confirmed whether the somatic embryos were generated from origins other than callus cells and whether somatic variation was suppressed during the induction culture. The present article is concerned with the quality of this system as a propagation system. We carried out histological observation and analysis of RAPD in two-step DSE using carrot hypocotyls.
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2. Materials and methods
2.2. Histological observation
2.1. Cells and medium
Morphological development in the course of the cultures was observed under a stereo microscope equipped with a digital camera on the torinocular port. Thin-sectioned segment samples were observed under a differential interference microscope for histological observation. Samples used for thin sectioning were fixed with 7.2% formaldehyde in 50 mM sodium phosphate buffer with 0.1% IGEPAL CA-630 (MP Biomedicals, Inc.) and 10% dimethyl sulfoxide for 40 min at room temperature. They were embedded in O.C.T. (Optical Cutting Temperature) compound (Tissue Tek) and frozen in liquid nitrogen. Frozen sections of 5-m thickness were prepared using a cryostat (Leica CM1850, Nussloch, Germany). The sections were placed on slide glasses shredded with 3% low-melting temperature agarose. The segments were fixed with a mixture of 38% formaldehyde solution, acetic acid and 70% ethanol (1:1:18), and freeze sec-
Seeds of carrot (Daucus carota L. cv. Natsumaki-Senkougosun) were surface-sterilized by soaking in 70% (v/v) ethanol for 3 min and then in 1% active sodium hypochlorite solution for 30 min. They were washed extensively in sterile water and then allowed to germinate for 1–3 weeks at 26 ◦ C in darkness on Murashige and Skoog (MS) solid medium with 8 g L−1 of agar without sucrose. Hypocotyl explants without apical meristems were removed from the seedlings and cut into 0.5 cm segments. The hypocotyl segments were placed on MS solid medium with 0.5 mg L−1 of 2,4-D, 30 g L−1 of sucrose and 8 g L−1 of agar. They were incubated at 26 ◦ C under continuous light of 2000 Lx (induction culture). After 14 days, the segments were transferred to new MS solid medium without 2,4-D and incubated for another 30 days (development culture).
Fig. 1. Typical morphological change in a hypocotyl segment during two-step DSE. A segment was incubated for 44 days, which included the first 14 days of the induction culture and the following 30 days of the development culture. The culture periods of the shown segment were 0 days (a), 7 days (b), 14 days (c), 21 days (d), 25 days (e) and 28 days (f). The numbers in (d) represent the indices for estimating the dependency of somatic embryogenesis on the location in a segment, as shown in Fig. 2.
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tions were prepared for propidium iodide (PI) staining. Twenty microliter of PI working solution, which contained 5 mg L−1 of PI in PBS buffer, was dropped on the frozen section [14]. The section was incubated at room temperature for 10 min, rinsed with PBS buffer and observed under a confocal laser microscope (Olympus FV300, Tokyo, Japan). 2.3. Estimation of genetic stability by RAPD-PCR The genetic stability of the regenerated plants was estimated using RAPD-PCR. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen K.K.) according to the manufacturer’s protocol from hypocotyl segments after cutting, from segments after 2 weeks of induction culture, from regenerated plantlets after a further 4 weeks of developmental culture, and from callus-forming segments after 6 weeks of induction culture. A set of these samples was prepared from one seedling. Their RAPD patterns were compared using four sets of samples. RAPD-PCR was carried out using the Ready-To-Go RAPD Analysis Kit (Amersham Pharmacia Biotech) according to the
Fig. 2. Dependency of somatic embryogenesis on the location in a donor segment. The numbers of regenerated plantlets were counted in each location of a segment that was fractioned and numbered from the root side as shown in Fig. 1(d). The ratios of regenerated plantlets in each location to the total count were evaluated based on the observation of four hypocotyl segments.
Fig. 3. Cross-sections of hypocotyls before cultivation (a and b) and after 14 days of induction culture (c and d). The donor hypocotyls were 3 cm (a and c) and 6 cm (b and d) in length. Abbreviations: v, vascular tissue; co, cortex; ep, epidermis; cav, cavity.
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manufacturer’s protocol. Five primers including the kit (No. 1–5) were used. The condition for the PCR was 95 ◦ C for 5 min (initial melting) and 45 cycles of 95 ◦ C for 60 s, 36 ◦ C for 60 s, and 72 ◦ C for 120 s. The PCR products were electrophoresed on 3.0% agarose gels (Nusieve 3:1 Agarose) and were stained with ethidium bromide. 3. Results and discussion 3.1. Morphological observation of somatic embryogenesis Fig. 1 shows a series of typical morphological changes in a hypocotyl segment during two-step DSE. The segment expanded during the first week (a and b). After 14 days (c), the epidermal tissue became transparent and the vascular tissue was yellowed. The edges of the segment expanded and yellowed. The segment was transferred to the development medium on the 14th day. After 25 days (11 days in the development culture) somatic embryos were generated from under the transparent epidermal cells (e). Plantlets from the somatic embryos developed and covered the donor segments in a 44-day culture (30-day development culture). The plantlets were 1–3 cm in length and could be used as donors for the next direct somatic embryogenesis. The dependency of somatic embryogenesis on the location in a donor segment was illustrated in Fig. 2. The number of plantlets appearing on photograph was counted after 11 days of development culture. The average total number of plantlets from a segment was 24. At the end of the culture, 4–5 times more plantlets were observed than at the 11th day of the development culture. However, the origin of the plantlets could not be determined at the end of the cultures. Callus formed on each edge of the segment and somatic embryos were generated from the whole segment except for the callus part. A high frequency of regenerated somatic embryos was observed near one edge of the segments. The edge did not, however, depend on the polarity of the donor plants. Therefore the deviation for the number of somatic embryos generated from the ends of the segments was bigger than those for other data. Shortening the donor segments to 0.2 cm did not influence the frequency of somatic embryogenesis (data not shown). 3.2. Histological observation
Fig. 4. Thin section of hypocotyl segment in the development culture with a thick cell wall structure. The structure was observed at the outer part next to the epidermis on the 5th day of the development culture (a). This structure was observed to be fibrous in axial section. Somatic embryos were observed on the structure at the 7th day of development culture (b). Abbreviations: v, vascular tissue; co, cortex; ep, epidermis; tw, structure with a thick cell wall; se, somatic embryo.
Histological observation was carried out to determine the location from which somatic embryos were generated. Two groups of donor hypocotyls with different lengths were compared. We found that the number of somatic embryos generated from a segment decreased as the hypocotyl length of the seedling increased [7]. Fig. 3(a–d) shows cross-sections of two of the donor hypocotyls before cultivation and after 14 days of induction culture. These hypocotyls were 3 and 6 cm in length. Cortex with parenchyma cells filled between the epidermis and vascular tissue. The number of parenchyma cells was smaller in the longer hypocotyl than the shorter one before the culture (Figs. 3a, 3b).
After 14 days, a cavity was observed in the longer hypocotyl (Fig. 3d). A structure with a thick cell wall was formed at the outer part next to the epidermis on the 5th day in the development culture as shown in Fig. 4a. This structure was observed to be fibrous in the axial section. The formations of this structure were observed in all segments from the 3-cm hypocotyls, although the time to form the structure varied with the individual hypocotyl from the 10th day of the induction culture to the 5th day of the development culture. And it could not be observed in segments from 6-cm hypocotyls. Somatic embryos were observed on the
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Fig. 5. Cross-section of a segment stained with propidium iodide after 10 days of development culture. Fluorescence was observed in the somatic embryos and in the structure with a thick cell wall. Abbreviations: v, vascular tissue; ep, epidermis; tw, structure with a thick cell wall; se, somatic embryo.
fibrous structure on the 7th day of the development culture as shown in Fig. 4b. Fig. 5 showed a cross-section of a segment after 10 days of development culture staining with PI. The fluorescence of PI, which stains nucleotides, was observed in the somatic embryos and in the structure with a thick cell wall. From these observations, this structure was concluded to be similar to collenchyma, because it was fibrous, surrounded by a thick cell wall, and contained rich nucleotides. This collenchyma-like structure is necessary to generate somatic embryos, judging from the results showing that the somatic embryos were generated from the structure and the structure was not observed in 6-cm-long hypocotyls which had a low potential to generate somatic embryos. The epidermal cells may differentiate and prolong along with the development of the hypocotyls. The growth of the parenchyma cells may not be able to catch up with the increase in the length. Thus, the density of the parenchyma cells may decrease in the longer hypocotyls. We think that the parenchyma cells with low density would part from the epidermis during the induction culture and that a cavity would be generated. The
Fig. 6. A representative banding pattern of RAPD-PCR products using template DNAs from donor hypocotyls (D), induction-cultured segments (I), regenerated plantlets (R) and callus-forming segments (C). Two PCR products (arrow) disappeared with the callus-forming segments.
parenchyma cells that are separated from the epidermis might not be able to form the fibrous structure. Masuda et al. reported that somatic embryos were generated directly from the epidermal cells of donor explants when the shorter treatment with 2,4-D was used [8]. In our two-step DSE, somatic embryos were generated from the secondary formed collenchyma-like structure. This observation locates the twostep DSE between the direct somatic embryogenesis and the callus-mediated somatic embryogenesis. 3.3. Analysis of somaclonal variation The genetic stability was evaluated with RAPD-PCR. The PCR was carried out using the four sets of hypocotyls with five primers. The number of amplified DNA fragments, which ranged in size from 300 to 1500 bp, ranged from 4 to 12 depending on the primer that was used. Fig. 6 shows a rep-
Table 1 Observed number of PCR products
Total number of PCR products from donor hypocotyls Number of disappeared bandsa Number of emerged bandsa Mutation index (%)b a b
Induction cultured segments
Regenerated plantlets
0 0 0.0
1 0 0.6
Callus forming segments
169
The number of disappeared and emerged bands compared to the donor hypocotyls. Mutation index was the ratio of disappeared and emerged bands to the observed PCR products with donor hypocotyls.
21 0 12.4
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resentative banding pattern of PCR products amplified using template DNAs from a donor hypocotyl, an induction-cultured segment, a regenerated plantlet and a callus-forming segment. Table 1 summarizes the observed number of PCR products and the number of disappeared and emerged bands in comparison to the donor hypocotyls. A mutation index was defined as the ratio of disappeared and emerged bands to the observed PCR products with the donor hypocotyls. The PCR products counted 169 with the donor hypocotyls. Only 1 product disappeared with the regenerated plantlets, and 21 products disappeared with the callus-forming segments. No change was observed with the induction-cultured segments. No emerged products were observed in all cases. The mutation indexes were 0, 0.6 and 12% for the inductioncultured segments, regenerated plantlets, and callus-forming segments, respectively. Therefore, the plantlets induced with two-step DSE will have higher genetic stability than callus cells. 4. Conclusion We characterized two-step DSE using carrot hypocotyls. RAPD-PCR research revealed that this method has genetic stability similar to other direct somatic embryogenesis systems. This genetic stability of the regenerated plantlets was supposed to be related to the origin of the somatic embryos. The somatic embryos were generated from the secondary structure, a collenchyma-like structure, rather than from callus cells as observed in this study. Furthermore, somatic embryogenesis from the secondary structure may have caused the frequency of regeneration to be greater than that of one-step direct somatic embryogenesis and other organogenesis systems. Acknowledgments This research was supported in part by a Grant-in-Aid for Scientific Research (No. 12750707) and a Grant-in-Aid for Advanced Scientific Research on Bioscience/Biotechnology Areas from the Ministry of Education, Science, Sports and Culture of Japan.
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