Journal of Experimental Botany, Vol. 53, No. 374, pp. 1575-1580,
July 1, 2002
© 2002 Oxford University Press
Establishment of a reproducible tissue culture system for the induction of Arabidopsis somatic embryos
Received 13 November 2001; Accepted 20 March 2002
Gene Research Center, Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Abbreviations: ABA, abscisic acid; 2,4-D, 2,4 dichlorophenoxyacetic acid.
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Abstract |
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Somatic embryogenesis is an example of totipotency and is used as a model system for studying embryogenesis. A reproducible tissue culture system was established for the large-scale induction of Arabidopsis somatic embryos. The method allows maintenance of high embryogenic competence over a one-year period. Using this tissue culture system, the expression of embryo-specific genes (ABI3, LEC1, FUS3) was detected in embryogenic cells and somatic embryos. Exogenous application of abscisic acid enhanced the expression of some late-embryogenesis-abundant (LEA) protein genes in somatic embryos. The experiments show that the method can be used to obtain sufficient amounts of embryogenic material for basic molecular analyses.
Key words: Key words: Arabidopsis thaliana, embryo-specific gene expression, somatic embryogenesis, zygotic embryos.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Arabidopsis is extensively used as a model plant for genetic and molecular biological studies of various phenomena in plants. Embryogenesis is one of the most important steps in plant development, and many Arabidopsis mutations and genes that relate to embryogenesis have been isolated and analysed (Meinke, 1995). However, a detailed analysis of embryogenesis at the protein level has been hampered by difficulties in isolating and cultivating a sufficient number of immature, early-stage zygotic embryos.
Instead of analysing zygotic embryos, carrot somatic embryos (SEs) have been used as experimental models for the elucidation of physiological, biochemical and molecular biological events during embryogenesis (Zimmerman, 1993). Indeed, various reports have indicated that SEs are morphologically and physiologically similar to zygotic embryos (Higashi et al., 1998; Shiota et al., 1998, 1999).
Somatic embryogenesis in Arabidopsis has been induced from immature zygotic embryos (Sangwan et al., 1992; Wu et al., 1992) and protoplasts of leaf-derived cells (O’Neill and Matthias, 1993; Luo and Koop, 1997). However, only a limited number of SEs were obtained in these culture systems. Pillon et al. (1996) reported a modified culture system, in which embryogenic cells (ECs) were induced from primary SEs obtained from immature zygotic embryos, and secondary SEs were formed from the ECs. Several claims have been made recently that some proteins such as arabinogalactan proteins and receptor kinase were involved in embryogenesis. These reports suggested that either over-expression or repression of the protein genes could have resulted in the induction of somatic embryos (Mordhorst et al., 1998; Shah et al., 2001a, b; van Hengel et al., 2001; Hecht et al., 2001). However, these systems did not provide large quantities of induced SEs in the Arabidopsis ecotypes used, because the embryogenic cell proliferation rate was low and embryogenic competence was rapidly lost.
This communication reports on a reproducible tissue culture system for large-scale induction of Arabidopsis SEs and techniques for maintaining high embryogenic competence over long periods of time. Moreover, the embryos were examined for the expression of embryo-specific genes in vitro in order to validate the suitability of the system to study embryogenesis.
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Materials and methods |
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Plants and primary SE induction
Arabidopsis thaliana (L.) Heynh. (Columbia) plants were grown at 21 °C under conditions of continuous light (white light at 50 µmol photons m–2 s–1) for about 8–12 weeks. At the end of the growth period, young green siliques were collected, surface-sterilized with 1% NaClO solution for 15 min, and washed with sterile distilled water. The siliques were dissected under sterile conditions using a dissecting microscope. Immature seeds were selected from the siliques, and immature zygotic embryos were isolated. These were placed on agar-solidified B5 medium containing 4.5 µM 2,4-D (Pillon et al., 1996). Primary SEs formed after 8–21 d. The primary SEs were transferred to liquid B5 medium containing 9.0 µM 2,4-D in order to induce embryogenic cell clusters (ECs), which were composed of embryogenic cells and secondary SEs. ECs were subcultured every 2 weeks in B5 medium containing 9.0 µM 2,4-D. To induce morphologically differentiated SEs, 2-week-old cultures of EC were washed five times with phytohormone-free liquid B5 medium and transferred to phytohormone-free liquid B5 medium.
Re-induction of ECs from differentiated SEs
The ECs lost the ability to transform into SEs during repeated subculturing in liquid B5 medium containing 9.0 µM 2,4-D. The ECs were transferred to solid B5 medium containing 4.5 µM 2,4-D and cultivated under conditions of continuous light (white light at 50 µmol photons m–2 s–1) at 21 °C. Green SEs formed after about 10 d and were transferred to liquid B5 medium containing 9.0 µM 2,4-D. The green SEs developed into ECs once more after 14 d of incubation. SEs were derived from these ECs by the method described above.
RNA isolation and Northern blotting
Total RNA was isolated using the phenol/SDS method (Ausubel et al., 1987). Total RNA (10 µg lane–1) was loaded onto a 1.2% agarose gel containing 2.2 M formaldehyde, subjected to electrophoresis and transferred to a Biodyne B nylon filter (Pall BioSupport, East Hills, NY, USA). The filters were hybridized at 42 °C for 20 h with 32P-labelled DNA fragments from ABI3 [nucleotides (nt) 750–1489], LEC1 (nt 17–689), FUS3 (nt 1–1153), ATECP31 (nt 1–1080), and ATECP63 (nt 1–1600), in a hybridization solution that contained 50% formamide, 5x Denhardt’s solution, 6x SSPE, 0.5% SDS, and 150 µg herring sperm DNA.
Construction of the Arabidopsis vector
An ABI3 promoter fragment (nt –872 to +25) with HindIII and BamHI sites at the 5'- and 3'-ends, respectively, was PCR amplified using Arabidopsis genomic DNA as the template. The PCR product was cloned into the HindIII and BamHI sites in pBluescript II SK+ (Stratagene) and the recombinant plasmid was confirmed by DNA sequencing. This fragment was further sub-cloned into the HindIII and BamHI sites (upstream of the GUS gene) in the pBI101 binary vector and subsequently transferred into Agrobacterium tumefaciens strain C58C1Rifr (PGV2260) by triparental mating (Bevan, 1984).
Arabidopsis transformation and GUS (ß-glucoronidase) assays
The transformation of Arabidopsis thaliana (Columbia) was performed as described by Bechtold et al. (1993). Histochemical GUS assays were carried out according to a previously reported method (Jefferson, 1987). Transgenic Arabidopsis tissues were dipped in a staining solution containing 1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-ß-glucuronide), 50 mM NaH2PO4 and 0.1% Tween 20. The samples were incubated overnight at 37 °C.
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Results |
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A tissue culture system for the induction of Arabidopsis somatic embryos
Immature zygotic embryos at various stages of development were isolated from green siliques and transferred onto B5 agar medium containing 4.5 µM 2,4-D. Some green SEs (primary SEs) were induced on the surfaces of the explants after 10 d of culture in light (Fig. 1B). When bent-cotyledon embryos (Fig. 1A) were used as explants, around 90% of the explants formed SEs. On the other hand, when zygotic embryos at earlier developmental stages (e.g. heart-shaped, torpedo-shaped and walking-stick-shaped stages) were used, the frequency of embryo formation was low (under 60%). Most of the primary SEs showed morphological abnormalities, such as fused cotyledons and fused multiple embryos (Fig. 1B). When the green primary SEs were excised and cultured in B5 liquid medium containing 9.0 µM 2,4-D in darkness, EC clusters that consisted of undifferentiated cells and large numbers of immature SEs were obtained (Fig. 1C). About 0.02 g of SEs were obtained per 10 ml of culture, approximately 2 weeks after transfer of the ECs to phytohormone-free medium (Fig. 1D). These secondary SEs showed normal morphology, i.e. contained a radicle, a hypocotyl, and two cotyledons (Fig. 1E).
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Maintenance of high embryogenic competence
ECs were subcultured every 2 weeks for approximately 2 months in B5 liquid medium containing 9.0 µM 2,4-D. During this period, the ECs assumed a watery consistency and their colour changed from yellowish to clear (Fig. 1F). Embryogenic competence decreased dramatically with successive passages in culture and eventually no SEs formed, even after transfer of the ECs to phytohormone-free B5 liquid medium. In this case, the ECs formed multiple adventitious roots rather than SEs. On the other hand, secondary green SEs formed when ECs with decreased embryogenic competence were cultured on solid B5 medium containing 4.5 µM 2,4-D in light (Fig. 1G). The green SEs resembled primary SEs formed from immature zygotic embryos, and new ECs could be induced from the secondary green SEs using the culture method described above. High embryogenic competence could be restored repeatedly using this method.
Expression of embryo-specific genes in vitro
The expression of embryo-specific genes was examined using primary SEs, ECs, secondary SEs, and zygotic embryos. The expression of ABI3 (Giraudat et al., 1992), FUS3 (Luerßen et al., 1998) and LEC1 (Lotan et al., 1998) was confirmed by Northern blotting in primary SEs, ECs and secondary SEs induced from ECs, as well as in young siliques containing zygotic embryos, but not in flower buds or stems (Fig. 2). The expression levels of ABI3 and FUS3 were significantly higher in SEs and ECs than in siliques. Enhanced expression of these genes in SEs was not induced by the addition of ABA to the cultures.
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The expression of AtECP31 (Yang et al., 1996) and AtECP63 (Yang et al., 1997), which are typical LEA proteins, was observed in green siliques containing developing zygotic embryos, but not in leaves and young seedlings (Fig. 3). When AtECP31 was used as a probe, expression was not observed in SEs and ECs in the absence of ABA, but was detected in SEs that had been treated with ABA (Fig. 3). AtECP63 expression was observed in primary SEs, ECs and secondary SEs, as well as in green siliques containing developing zygotic embryos. AtECP63 expression was enhanced by ABA treatment of SEs (Fig. 3).
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Histochemical GUS assay on ABI3 pro::GUS transgenic plants
Histochemical GUS assays were carried out on ABI3 pro::GUS transgenic plants, in which GUS expression was controlled by the ABI3 gene promoter. GUS expression (as indicated by the appearance of a blue colour in stained sections) was not observed in leaves, flower buds or roots of adult plants (Fig. 4A). However, GUS expression was observed in seeds, and especially in zygotic embryos at various developmental stages (Fig. 4B, C). GUS expression was observed in both SEs and ECs that were directly induced from ABI3 pro::GUS transgenic plants by the method described above (Fig. 4D).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Wu et al. (1992), Sangwan et al. (1992) and Luo and Koop (1997) reported the formation of SEs from immature zygotic embryos of several different Arabidopsis ecotypes. However, the limited number of SEs obtained in these cultures made it difficult to pursue biochemical and molecular biological analyses. Pillon et al. (1996) reported that ECs in the ecotype Columbia could be induced from primary SEs formed on immature zygotic embryos, and that secondary SEs could be generated from ECs. Despite the establishment of an SE-induction system, the number of SEs obtained was still insufficient for biochemical and molecular biological analyses, and the SEs showed abnormal morphological features, such as fused cotyledons and multiple fused embryos (Fig. 1B). Therefore, an attempt was made to modify the previously developed method of Pillon et al. (1996) using the ecotype Columbia.
In the Pillon et al. (1996) method, ECs were induced on solid B5 medium. However, it was not possible to get enough ECs to proliferate using this method, probably because ECs divide slowly on solid media. Liquid culture media were also tested for the induction and maintenance of ECs (Fig. 1C). Sufficient quantities of ECs and a large quantity of SEs from ECs were obtained by using liquid cultures. Moreover, the secondary SEs had normal morphology (Fig. 1E). In this culture system, light was not a requirement for the induction of ECs and SEs.
On the other hand, it is generally known that the embryogenic competence of ECs decreases with successive subcultures. Pillon et al. (1996) reported that Arabidopsis ECs grown on solid medium maintained embryogenic competence for over 1 year. However, in these experiments, ECs grown in liquid cultures lost embryogenic competence more rapidly than those grown on solid media (Fig. 1F). It is likely that the high proliferation rate of ECs in liquid culture leads to the dramatic decrease in embryogenic competence. Thus, it was necessary to develop a method for maintaining high embryogenic competence over long periods of time.
SEs could not be induced from ECs that had low embryogenic competence in phytohormone-free B5 medium. The ECs formed multiple roots in both phytohormone-free liquid and solid media. However, it was possible to induce SEs from ECs with low embryogenic competence by growing the ECs in light on solid B5 medium containing 4.5 µM 2,4-D (Fig. 1G). In this case, the provision of light was the most important factor. The green SEs induced from ECs with low embryogenic competence were quite similar to the primary SEs formed from zygotic embryos. A new EC line could be induced from the green SEs. Using this method, ECs could be maintained in a state of high embryogenic competence for over 1 year.
In the carrot somatic embryogenesis system, the expression of some embryo-specific genes was reported in SEs and ECs, as well as in zygotic embryos (Shiota et al., 1998). In Arabidopsis, some embryo-specific genes, such as ABI3, LEC1 and FUS3, have been reported (Giraudat et al., 1992; Lotan et al., 1998; Luerßen et al., 1998). Gene expression has been observed in zygotic embryos and starts during the early stages of embryo development (Lotan et al., 1998; Luerßen et al., 1998). The expression patterns of these genes were examined in the SE-induction culture system. The expression of ABI3, LEC1 and FUS3 was observed in zygotic embryos, SEs and ECs, but not in vegetative tissues (Fig. 2). Moreover, GUS expression driven by the ABI3 gene promoter was observed in zygotic embryos and SEs (Fig. 4D). These results show that embryo-specific gene expression in SEs is similar to that in zygotic embryos, and suggest that the developmental programmes in SEs are similar to those in zygotic embryos.
It is well known that ABA induces the expression of LEA (late-embryogenesis abundant) genes in late-stage zygotic embryos. The expression of some carrot LEA genes was observed in SEs that had been treated with ABA (Shiota et al., 1998). Therefore, the expression of Arabidopsis LEA genes was examined in the culture system. AtECP31 (Yang et al., 1996) and AtECP63 (Yang et al., 1997) represent Arabidopsis LEA genes that are homologous to the carrot LEA genes, such as ECP31 and ECP63, whose expression is induced by ABA via ABI3-mediated signal transduction (Shiota et al., 1998). The expression of AtECP31 and AtECP63 was induced by ABA treatment of Arabidopsis SEs. This result and the observed expression of ABI3 in SEs indicate that ABA-mediated signal transduction operates in both SEs and zygotic embryos of Arabidopsis.
In summary, this paper communicates the refinement of previously published methods of tissue culture which allows a reproducible tissue culture system for the induction of SEs in Arabidopsis to be established. Using this method, large quantities of SEs and ECs were obtained. Moreover, the SEs induced in this culture system resembled zygotic embryos, both morphologically and functionally. The culture system reported here provides a useful tool for elucidating physiological, biochemical and molecular biological events during embryogenesis, and an alternative to the use of zygotic embryos.
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Acknowledgements |
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This work was supported in part by a Grant-in-Aid for ‘Research for the future’ program from the Japan Society for the Promotion of Science (JSPS-RFTF00L01601), by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and by the Special Coordination Funds from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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