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Development and Morphogenesis |
Department of Plant Cellular and Molecular Biology, The Ohio State University, 318 West 12th Avenue, Columbus, Ohio 43210 USA
Received for publication February 12, 2004. Accepted for publication July 30, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Arabidopsis thaliana • callus initiation • cell cycling • cell expansion • 2,4-dichlorophenoxy acetic acid • embryo culture • somatic embryogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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who showed that a callus produced from any vegetative part of carrot (Daucus carota) such as the root, petiole, or inflorescence stalk reared in a medium containing a high concentration of 2,4-D formed somatic embryos upon transfer to a medium with a reduced level of the auxin. From this time onwards, the use of a defined medium and a single-step transfer of a callus or a cell suspension growing in a medium supplemented with a moderate dose of 2,4-D to one containing a reduced amount of the auxin or none at all, was adopted as the standard protocol to study the physiology and molecular biology of somatic embryogenesis in carrot and became widely popular in inducing somatic embryogenesis in a broad range of species (Thorpe and Stasolla, 2001
; Raghavan, 2004
). However, the precise role of 2,4-D in somatic embryogenesis in carrot, especially whether the cells are programmed for embryogenesis before they encounter auxin in the medium or after, has not been established (Dudits et al., 1995
; Raghavan, 1997
; Chugh and Khurana, 2002
).
The use of 2,4-D alone or in combination with other hormones has become almost routine in inducing somatic embryogenesis in seed cultures (Huang and Yeoman, 1984
; Mordhorst et al., 1998
) and in cultured zygotic embryos of the model plant Arabidopsis thaliana (Sangwan et al., 1992
; Wu et al., 1992
; Pillon et al., 1996
; Luo and Koop, 1997
; Mordhorst et al., 1998
, 2002
; Ikeda-Iwai et al., 2002
). In the protocol developed by Pillon et al. (1996)
using 2,4-D as the sole hormone, heart-shaped to walking-stick-shaped embryos were initially cultured in a liquid medium containing 4.5 µmol/L 2,4-D for 21 d to induce the formation of early-stage somatic embryos followed by their transfer to an auxin-free medium for plantlet formation. This work also showed that it was possible to obtain cell lines with continued embryogenic potential if early-stage somatic embryos were maintained on a solid medium with an increased concentration of the auxin; this observation formed the basis of the protocol developed by Ikeda-Iwai et al. (2002)
. In a modification of the Pillon et al. (1996)
method, Mordhorst et al. (1998
, 2002
) cultured bent-cotyledon stage embryos of wild-type A. thaliana and of mutants with defective shoot apical meristem such as shoot meristemless (stm), wuschel (wus), and zwille/pinhead (zll/pnh) in a liquid medium containing 4.5 µmol/L 2,4-D for 21 d, followed by subculture of specially selected green, embryogenic cell clusters in an auxin-free solid medium for the development of somatic embryos. Although these studies showed that reprogramming of cells of cultured zygotic embryos by 2,4-D is a critical first step in the induction of somatic embryos, the signal transduction pathway triggered by auxin in this system has not been determined. According to Zuo et al. (2002)
, overexpression of a WUS-related gene, PLANT GROWTH ACTIVATOR (PGA), induces high-frequency somatic embryogenesis in vegetative tissues and zygotic embryos of Arabidopsis in a hormone-independent way. It has been suggested from this work that the WUS/PGA gene modulates somatic embryogenesis by promoting embryogenic transition of somatic cells and/or by maintaining their embryogenic competence. Harding et al. (2003)
have recently shown that expression of the MADS-box gene AGAMOUS-Like15 (AGL15) also enhances the production of somatic embryos on zygotic embryos of Arabidopsis cultured in a hormone-free mineral salt medium.
In view of the potential use of somatic embryos as a model for zygotic embryos in biochemical and molecular investigations, much of the previous work on somatic embryogenesis in Arabidopsis was concerned with increasing the yield of embryos from cultured explants without interference by organogenesis. This has also resulted in the successful demonstration of enhanced somatic embryogenesis in seedling cultures of mutants such as primordial timing (pt) (allelic to altered meristem program1 [amp1], constitutive photomorphogenic2 [cop2] and häuptling [hpt]), clavata1 (clv1), and clv3 with enlarged shoot apical meristem (Mordhorst et al., 1998
; von Recklinghausen et al., 2000
), and of the shift in fate leading to somatic embryogenesis from vegetative parts of plants expressing the LEAFY COTYLEDONI (LEC1) (Lotan et al., 1998
) and LEC2 genes (Stone et al., 2001
). Consequently, the advantages inherent in a population of 15 000–20 000 cells of the zygotic embryo (Jürgens and Mayer, 1994
) undergoing morphogenetic changes in response to 2,4-D within a relatively short time have not been exploited to gain insight into the origin of somatic embryos and the basis for the acquisition of embryogenic competence by somatic cells. Using a transgenic line of Arabidopsis harboring a cyclin1 At:ß-glucuronidase (GUS) reporter gene construct, the present study has examined the spatial and temporal patterns of cell expansion, cell cycling, and morphogenesis during continuous culture of zygotic embryos in a medium containing 2,4-D. The goal of this study is to evaluate those changes that lead to embryogenic development of somatic cells due to 2,4-D action in specific parts of the embryo and to provide baseline histogenetic data for characterizing the role of the auxin in initiating gene activity and cell signaling for the induction of somatic embryos.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Tissue culture and cytology
For embryo culture, siliques enclosing ovules at the bent-cotyledon-stage embryo were harvested from the primary inflorescence axes of plants. They were surface-sterilized for 10 min in 12% chlorine bleach and washed three times in sterile distilled water. Siliques were opened in a few drops of water with fine forceps and microscalpel under a dissecting microscope, and embryos were squeezed from ovules with gentle pressure. They were immediately transferred with forceps to 60-mm diameter glass petri dishes containing 10 mL of liquid medium containing major salts of the B5 formulation of Gamborg (1975)
and minor salts according to Murashige and Skoog (1962)
, supplemented with 2% sucrose and 4.5 µmol/L 2,4-D, at pH 5.8–6.0. Petri dishes, each containing 10-15 embryos, were sealed with parafilm and were kept on a rotary shaker (80 rpm) at 25°C in continuous light provided by two fluorescent tubes (6 µphotons/µmole). Embryos were subcultured in a fresh supply of the same medium once at 10 d during a 20-d experimental period or transferred to the basal medium without 2,4-D at intervals of 2 d beginning at subculture. For the latter purpose, embryos were washed five times with the liquid basal medium and were placed on the surface of 10 mL of solidified medium contained in 60-mm diameter plastic petri dishes, at 5–7 embryos per dish. Cultures were maintained in an incubator at 25°C in complete darkness for 14 d. Gross morphogenetic changes in the embryos were monitored at 2-d intervals by examining cultures in a Zeiss inverted microscope and/or by photographing them in a Wild dissecting microscope using Kodak Technical Pan film.
Immediately after excision and at 1-d intervals during the first 7 d of culture in the liquid medium, growth measurements of about 30 embryos were made using a microscope fitted with an ocular-micrometer. The root-hypocotyl axis was measured from the extreme apex of the root to the base of the cotyledons. Cotyledons were measured from the base of their origin at the shoot apical meristem to the tip. Because the two cotyledons of an embryo grew in length somewhat unequally, measurements of the cotyledon that grew maximally in each embryo were used. The widest region of the hypocotyl was measured to determine its width. For histological studies, embryos collected immediately after isolation and at 1-d intervals during a 10-d experimental period were fixed in acetic alcohol, dehydrated in ethanol, n-propanol, and n-butanol series and embedded in glycol methacrylate following standard procedures (Feder and O'Brien, 1968
). Serial sections cut at 5–7 µm thickness on a rotary microtome equipped with a steel knife were double-stained in periodic acid-Schiff (PAS) and toluidine blue and mounted in Permount. Slides were examined under brightfield optics in a Zeiss Photomicroscope III, and sections were photographed using Kodak T-Max 100 film. Because preliminary observations showed that anatomical changes leading to somatic embryogenesis were initiated at the base of the cotyledons of cultured embryos, this region, within a length of 100 µm from the origin of the cotyledons at the shoot apical meristem, formed the focus of attention in anatomical studies. The width of the base of cotyledons was determined microscopically from longitudinal sections of embryos cut through the procambial strands of the hypocotyl and cotyledons. These same sections were also used to count the number of layers of procambial strands at the base of the cotyledons and of mesophyll cells on the adaxial (facing the shoot apical meristem) and abaxial (facing outside) sides at the base of the cotyledons (on either side of the procambium). The above measurements were made from sections of about 30 embryos from three experiments.
GUS staining and detection
GUS activity in cells of embryos immediately after isolation and at 1-d intervals after culture was detected histochemically using 5-bromo-4-chloro-3-indoly1-ß-D-glucuronide (X-Gluc; Sigma, St. Louis, Missouri, USA) as a substrate, according to the method described earlier (Raghavan, 2002
). Embryos were subsequently rinsed in water and cleared in Hoyer's solution (7.5 g gum Arabic, 5 mL glycerol, 100 g chloral hydrate, 30 mL water) (Vernon and Meinke, 1994
) and mounted immediately in the same solution to count the number of blue spots generated by the GUS reaction. Additional uncleared embryos were fixed at each time point in acetic alcohol and processed for microtomy as described. Sections were stained in PAS and mounted in Permount. Color photographs of whole mounts and sections of embryos were made using Fujichrome Sensia 100 or Kodak Elite Chrome 200 film.
All experiments described here were repeated several times with essentially the same results. Data were analyzed statistically according to Snedecor and Cochran (1967)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The cotyledons began to diverge in opposite directions in about 3 d after culture, eventually attaining a position perpendicular to the root-hypocotyl axis during the experimental period (Fig. 6; see also Fig. 12). As shown in Table 2, the width of the basal region of the cotyledons also began to increase beginning 1 d after culture. Although data are provided for changes in width of this part of the cotyledons of embryos for only the first 7 d of culture, the basal region and the remaining part of the cotyledons continued to increase in width during the entire experimental period. Histological observations showed that beginning at 2 d of culture, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons causing this region to bulge (Figs. 7–10). Occasionally, the procambial cells on the abaxial side at the base of the cotyledons and cells of the epidermis and mesophyll on both abaxial and adaxial sides in this region of the cotyledons also divided within 3–4 d of culture, but these divisions were initiated only after the procambium on the adaxial side has divided (Fig. 8). Characteristic of the procambium, the new cells produced were narrow and elongate; as these cells were displaced adaxially they enlarged and appeared densely staining, contributing to the increase in the number of cell layers in the basal region of the cotyledons (Table 2). The period of culture beginning at about 2 d appeared to be one of intense cell division activity as in some embryos, the epidermis at the base of the cotyledons broke loose to make room for the newly formed cells (Fig. 11). Moreover, continued divisions of cells in the newly cut-off procambium and its derivatives became increasingly random. For these reasons, it was difficult to make accurate counts of the number of cell layers in the basal part of the cotyledons of embryos beyond 4 d after culture. As seen in Table 2, beginning at 2 d after culture of embryos the number of layers of cells formed on the adaxial side at the base of the cotyledons was significantly higher than on the abaxial side. Cell divisions later spread to the abaxial side of the cotyledons, followed by extensive divisions throughout almost the entire length of both cotyledons to give rise to a callus of nonchlorophyllous parenchymatous cells overlaid by layers of densely chlorophyllous cells of the original mesophyll (Fig. 12). Simultaneous with the initiation of divisions in the procambial cells at the base of the cotyledons, division of cells of the procambial strands in the hypocotyl, accompanied by the formation of 4–6 additional layers of cells in the cortex was also noted in embryos sampled 2– 4 d after culture. No additional new cells were produced in the root-hypocotyl axis, which appeared to be dwarfed by the burgeoning growth of the cotyledons.
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; Luo and Koop, 1997
; Mordhorst et al., 1998
, 2002
; Zuo et al., 2002
).
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Cell cycling and cell expansion during somatic embryogenesis
To determine whether the anatomical and morphogenetic changes observed in cultured embryos are in agreement with the pattern of cell cycling, the spatial and temporal aspects of cell cycling in cultured embryos was monitored using the cyclin1At::GUS reporter gene construct in the wild-type transgenic plant. The fusion of CDB to the GUS gene in this construct leads to the degradation of CDBGUS protein at the end of mitosis, thus restricting the reporter gene expression to those cells passing through the mitotic cycle. These results need be interpreted with caution, because the method does not identify cells passing through a specific phase of the cell cycle such as the S-phase, which can be detected by autoradiography of 3H-thymidine labeling. Based on cleared whole mounts, a large number of histochemically detectable blue-staining cycling cells (42.3 ± 3.2 blue dots per embryo; N = 24) were distributed in both the root-hypocotyl axis and cotyledons of bent-cotyledon-stage embryos at the time of excision and culture (Fig. 21). There was a dramatic decrease in the number of GUS-expressing cells 1 d after culture (13.8 ± 1.2 blue dots per embryo; N = 23), followed by the appearance of new punctuate patches of blue-stained cells in the cotyledons after 2–3 d of culture (Figs. 22, 23). Cultured embryos initially displayed GUS reaction diffusely on the adaxial side at the base of the cotyledons, later spreading consistently as dots in a ring along the entire basal region of the cotyledons (Figs. 24, 25). Cycling cells were also observed occasionally in the root-hypocotyl axis and the shoot apical meristem (Fig. 25). In embryos cultured for 6–10 d, GUS-expression was confined to the margins of cotyledons associated with morphogenetic changes such as callus formation (Fig. 26) and appearance of globular (Fig. 27) and heart-shaped somatic embryos (Fig. 28) and disappeared more or less completely from other parts of the cotyledons.
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found that approximately 5% of GUS-accumulating cells are in mitosis and concluded that cyclin is expressed during G2 and mitosis. On the whole, the spatial and temporal patterns of expression of GUS-accumulating cells in cultured embryos are consistent with the anatomical and morphogenetic changes observed. In the absence of cycling cells in the root-hypocotyl axis during the first day of culture of embryos, it appears that the initial increase in length of this embryonic organ is due to the effects of 2,4-D on cell expansion. A significant increase in the width of the basal region of the cotyledons within 1 d after culture of embryos probably reflects an auxin effect on cell expansion in this part of the cotyledons preparatory to the formation of callus. As cycling cells are seen both in the hypocotyl and in the basal region of the cotyledons beginning 2 d after culture of embryos, it is reasonable to conclude that the production of new cells along with cell expansion contributes to the continued increase in length and width of the root-hypocotyl axis and to the increase in width of the basal region of the cotyledons during the next 5 d of culture when measurements were made.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 1988
; Vasil and Vasil, 1982
), legumes (Tetu et al., 1990
), woody plants (Chen et al., 1988
; Gupta et al., 1996
), and other assorted species (Williams and Maheswaran, 1986
) in which zygotic embryos constitute the starting material for the induction of somatic embryos. After a period of uncertainty about the favorable stage of the zygotic embryo of Arabidopsis for induction of somatic embryos in culture (Wu et al., 1992
; Pillon et al., 1996
; Luo and Koop, 1997
), the present work along with other previous investigations (Mordhorst et al., 1998
, 2002
; Ikeda-Iwai et al., 2002
) has now established that somatic embryos can be obtained reproducibly and in large numbers by the culture of bent-cotyledon-stage embryos in a medium containing 2,4-D. The ease of isolation of bent-cotyledon-stage embryos for culture makes the system particularly useful in future investigations on the cell and molecular biology of somatic embryogenesis in this plant.
Somatic embryogenesis in cultured zygotic embryos of Arabidopsis appears to involve phases of cell expansion, cell division, callus initiation, formation of early-stage embryos, and maturation of embryos. The phase of cell expansion, although brief, was clearly delimited by the absence of cycling cells and by the increase in width of the basal region of the cotyledons of embryos after 1 d of culture. Whereas the first four phases of the embryogenic sequence occur in the presence of 2,4-D in the medium, somatic embryos attain mature form when they are nurtured in a medium lacking auxin. In this respect Arabidopsis differs from the widely studied carrot somatic embryogenesis system in which 2,4-D induces the formation of unorganized cell clusters known as proembryogenic masses, a few cells of which form somatic embryos when transferred to a hormone-free medium (de Vries et al., 1988
; de Jong et al., 1993
). The results of the present work show that somatic embryogenesis in cultured zygotic embryos of Arabidopsis is achieved by auxin-induced division of cells of the cotyledons and that, as long as the cotyledons are in a mode of active divisions, other parts of the embryo such as the hypocotyl and shoot apical meristem do not participate in somatic embryogenesis. This is confirmed by the observation made in this work and by Luo and Koop (1997)
that culture of isolated cotyledons leads to the formation of a callus on which somatic embryos arise, just as cotyledons of cultured intact embryos. Moreover, explants consisting of other embryonic organs produce neither a callus nor somatic embryos. The initiation of divisions in the procambial cells of the cotyledons as the first of a series of events leading to somatic embryogenesis in cultured zygotic embryos of Arabidopsis finds a close parallel in the observations of Guzzo et al. (1994
, 1995
) that in the hypocotyl of carrot seedlings cultured in a medium containing 2,4-D, proembryogenic masses originate from the cells of the vascular cylinder, probably from the potential cells of the pericycle.
It has been claimed that embryogenic clusters or somatic embryos originate in cultured wild-type embryos of Arabidopsis from cells of the shoot apical meristem or from cells in the axils of cotyledons (Mordhorst et al., 2002
). The importance of the shoot apical meristem in somatic embryogenesis in Arabidopsis is also highlighted by the conclusion that increased somatic embryogenesis noted in seed cultures of pt and clv mutants is due to the abundance of undifferentiated meristematic cells in the shoot apical meristem to acquire embryogenic competence (Mordhorst et al., 1998
). The role of the shoot apical meristem in somatic embryogenesis, however, appears doubtful from the demonstration that somatic embryos are easily produced in cultured zygotic embryos of mutants of Arabidopsis impaired in the development of the shoot apical meristem, although cells in the cotyledonary axils of mutant embryos are believed to serve as the source of origin of somatic embryos (Mordhorst et al., 2002
). The formation of somatic embryos from vegetative parts of plants overexpressing the LEC and PGA genes also appears to question a requirement for the shoot apical meristem or undifferentiated meristematic cells for embryogenic induction (Lotan et al., 1998
; Stone et al., 2001
; Zuo et al., 2002
). Furthermore, results of some experiments on the frequency of somatic embryogenesis in AGL15 transgenic Arabidopsis plants in the pt background reported by Harding et al. (2003)
also do not support a relationship between the size of the shoot apical meristem and embryogenic competence. Anatomical studies made in the present work showing that divisions leading to the formation of a callus on which somatic embryos subsequently appear are initiated in the cotyledons beginning in the procambial cells on the basal adaxial side, as well as the observation that cultured embryo axes consisting of the shoot apical meristem and cells of the cotyledonary axils along with the root-hypocotyl axis do not initiate a callus, assign a major role for the cotyledons in callus initiation and somatic embryogenesis. Occurrence of the first divisions heralding callus growth on the basal adaxial side of the cotyledons probably suggests an indirect effect of the shoot apical meristem in the process.
Cyclin proteins expressed in the gene construct used in this study and other reporter gene constructs have served as molecular markers to analyze the role of cell cycling during the development of several organs and tissues of Arabidopsis, such as leaf blade and leaf veins (van Lijsebettens and Clarke, 1998
; Donnelly et al., 1999
; Kang and Dengler, 2002
), shoot and root apical meristems, lateral roots, floral organs (Ferreira et al., 1994
), and embryos (Ferreira et al., 1994
; Raz et al., 2001
; Raghavan, 2002
). The present work using the cyclin:: ß-glucuronidase reporter construct has reinforced anatomical observations showing that the formation of callus in cultured embryos of Arabidopsis is correlated with cell cycling in the adaxial side of the cotyledons and that the subsequent origin of somatic embryos initially involves cycling of cells at the margins of the callus. Although anatomical studies assigned the cells derived from the procambium a major role in the burgeoning growth of the callus at the base of the cotyledons, use of the reporter gene construct has confirmed the cycling of cells of the epidermis and mesophyll in this morphogenetic event.
The precise origin of somatic embryos has been traced in a few cases, although the pathways of their origin are very varied. Somatic embryos have been reported to arise in the peripheral as well as deep-seated cells of the callus derived from floral buds of Ranunculus scleratus (Konar and Nataraja, 1969
) and stem explants of Tylophora indica (Rao and Narayanaswami, 1972
), stem epidermis of plantlets formed in callus cultures of R. sceleratus (Konar et al., 1972
), superficial cells of proembryogenic masses in carrot cell suspension cultures (McWilliam et al., 1974
), internal meristematic cotyledonary cells of embryos of Theobroma cocao (Pence et al., 1980
), subepidermal cells of the scutellum of immature embryos of Pennisetum americanum (Vasil and Vasil, 1982
), peripheral cells of the callus induced in young leaves of Saccharum officinarum (Ho and Vasil, 1983
), epidermal cells of the hypocotyl and cotyledons of Medicago sativa embryos (Dos Santos et al., 1983
), epidermal and subepidermal cells of the scutellum and a callus of scutellar origin in immature embryos of Zea mays (Vasil et al., 1985
), epidermal cells of immature embryos of Trifolium repens (Maheswaran and Williams, 1985
), and scutellar epithelium (a layer of cells at the interface of the embryo with the endosperm) of embryos of Oryza sativa (Jones and Rost, 1989
). Although there is no unequivocal evidence for the single-celled origin of somatic embryos in Arabidopsis, in root explants overexpressing the PGA gene, single cells dividing transversely to give rise to globular embryos have been described (Zuo et al., 2002
). Pointing to the same conclusion, Luo and Koop (1997)
have found that culture of late heart-shaped-stage embryos of a Landsberg erecta ecotype of A. thaliana results in the formation of octant-to globular-stage somatic embryos with attached suspensor cells, very similar to their zygotic counterparts.
In summary, somatic embryogenesis in Arabidopsis induced by the continuous culture of bent-cotyledon-stage embryos in an auxin-containing medium followed by their transfer to an auxin-free medium provides a rapid and attractive system for studying gene activation during embryogenic transformation of somatic cells and the subsequent maturation of early-stage embryos without the complication of an intermediate stage of proembryogenic masses as in carrot. The ease and precision of response that have made Arabidopsis a model system for analysis of many developmental phenomena in plants also seem to be true in the case of somatic embryogenesis in this plant.
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FOOTNOTES |
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2 E-mail: raghavan.1{at}osu.edu
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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