Direct formation of heart-shaped embryos from differentiated
single carrot cells in culture
Hiroshi Yasuda, Masaaki Nakajima, Hiroshi Masuda *, Takuji Ohwada
Department of Agricultural Chemistry,Obihiro Uni6ersity of Agriculture and Veterinary Medicine,Obihiro080-8555,Hokkaido,Japan Received 19 May 1999; received in revised form 10 September 1999; accepted 10 September 1999
Abstract
Single cells of a variety of shapes and sizes, which had been released from regenerated carrot plantlets, divided with irregular patterns to form cell clusters of various shapes and sizes with uneven surfaces. Once these cell clusters had become isodiametric globular embryos with a smooth surface, they developed to heart-shaped embryos that were bilaterally symmetrical along the apical – basal axis. The transition of single cells to globular embryos was developmentally different from the transition of zygotes to globular embryos, which is caused by regular longitudinal and transverse divisions. Serial observations revealed a divergent sequence of morphological stages, from cell clusters to heart-shaped embryos during somatic embryogenesis. In some parts of cell clusters, globular embryos with cotyledonary primordia were formed, but cell clusters also developed directly to heart-shaped embryos without going through the typical globular stage. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Carrot; Morphogenesis; Somatic cell; Somatic embryogenesis
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1. Introduction
In dicotyledonous somatic embryogenesis, so-matic cells develop into plants via characteristic morphological stages, known as the globular, heart-shaped and torpedo stages, which closely resemble the stages of development of zygotic embryos [1]. Several aspects of somatic embryoge-nesis have been compared to those of zygotic embryogenesis in various species including bio-chemical resemblance [2] and microstructural and biochemical differences [3 – 6]. In the pathway from single somatic cells to globular embryos, the cell cluster is a stable multicellular intermediate. However, it is unclear whether the morphological development from single cells to globular embryos during somatic embryogenesis resembles that from
the fertilized egg to the globular embryo in zygotic embryogenesis [7]. Our experimental system [8,9], in which the transition from single somatic cells of carrot to globular embryos proceeds via cell clus-ters in one step in the absence of 2,4-D, seems to be suitable for clarification of this issue. Further-more, it allows us to study how somatic cells are converted to embryogenic cells since somatic cells released from carrot hypocotyls divided to directly yield cell clusters with embryogenic potential, without prior dedifferentiation. In a previous study [9], we showed that embryogenic somatic single cells, released from regenerated carrot plantlets, were significantly larger than those from conventional suspension cultures [10]. Our cells divided with variable patterns to form cell clusters of diverse shapes. These irregularly shaped cell clusters eventually developed to globular embryos that underwent isodiametric expansion to form spherical masses of cells. Thus, the features of our somatic embryo-forming system seem to be differ-ent from those of the fertilized egg cell-to-globular cluster transition that proceeds by transverse and Abbre6iations: 2,4-D, 2,4-dichlorophenoxyacetic acid; SEM,
scan-ning electron microscopy.
* Corresponding author. Tel.:+81-155-49-5551; fax:+ 81-155-49-5577.
E-mail address:[email protected] (H. Masuda)
asymmetric division in zygotes [11]. It is not clear how cell clusters with diverse shapes and uneven surfaces develop to globular embryos with a defin-ite and isodiametric shape, and these first morpho-genetic events in somatic embryogenesis are of particular interest.
In dicotyledonous plants, the early embryo is globular and then develops to a heart-shaped em-bryo that is bilaterally symmetrical along the api-cal – basal axis and has two cotyledons. An oblong embryo with a distinct growth pattern is observed between the globular and heart-shaped stages in carrot somatic embryogenesis [12]. The achieve-ment of bilateral symmetry from axial symmetry in plant embryogeny occurs at the time of the transition from the globular to the heart-shaped stage via the oblong stage.
In this report, we describe the formation of cell clusters of diverse shapes with variable patterns of division from single somatic cells, as well as the unusual sequence of morphological stages from cell clusters to heart-shaped embryos, in early somatic embryogenesis from cells derived from regenerated carrot plantlets.
2. Materials and methods
2.1. Preparation of regenerated plantlets
Regenerated plantlets were prepared as de-scribed in a previous paper [9]. In brief, surface sterilized carrot (Daucus carota L. cv. Koshingo-sun) seeds were germinated on solidified basal Murashige – Skoog (MS) medium [13]. Hypocotyl explants from seedlings were cultured in liquid MS medium with 2,4-D (1 mg/l) for 24 h and then cultured in 2,4-D-free medium. After 3 weeks of culture in 2,4-D-free medium, cell clusters that had formed were transferred to fresh MS medium. These cell clusters regenerated plantlets of 3 – 7 cm in length within 2 weeks of transfer to the fresh medium.
2.2. Somatic embryogenesis from regenerated plantlets
The procedure for somatic embryogenesis from regenerated plantlets was the same as that de-scribed previously [9]. Regenerated plantlets (20 – 30) were cultured in 20 ml of MS medium
containing 2,4-D (1 mg/l), for 24 h and then transferred to 2,4-D-free medium. Release of sin-gle cells from whole regenerated plantlets and formation of cell clusters from these single cells were apparent at 2 – 3 days and 3 – 4 days, respec-tively. Serial observations of the transition of sin-gle cells to cell clusters were made as follows. Single cells, released from regenerated plantlets into the medium 3 days after transfer to 2,4-D-free medium, were collected on nylon screens with 84 and 42 mm pores and then cultured in conditioned medium, which was the original culture medium after passage through the nylon screen with 42mm pores and further filtration through filter paper. Serial observations of cell clusters were made on material that had been collected from the culture 7 – 8 days after transfer to 2,4-D-free medium on mesh with 117 and 42 mm pores and then cultured in fresh medium.
2.3. Scanning electron microscopy
Samples were fixed in 2% glutaraldehyde at 4°C and then in 5% KMnO4, then dehydrated in a
graded ethanol series. Samples in 100% ethanol were then treated withtert-butyl alcohol and dried by the critical-point method. Samples for SEM were covered with a thin layer of gold and ob-served under a scanning electron microscope (JSM-63oiF; JS Nippon Denshi, Tokyo) operated at 5 kV.
3. Results and discussion
3.1. Serial obser6ations of the transition of single
cells to cell clusters
3.2. Serial obser6ations of the transition of cell
clusters to embryos
Halfmoon-shaped cell clusters were released from some indeterminate cell clusters, with subse-quent rounding to become isodiametric globular embryos (Fig. 2). As described in our previous paper [9], one cell cluster sometimes developed into several globular embryos. Although the pho-tographs in Fig. 3A, B and C, are not serial observations of the same cell cluster because the observations were made by scanning electron mi-croscopy, they demonstrate a transition from clus-ter to globular embryo via a proglobular stage. The surface of the developing cell cluster is irregu-lar and rough (Fig. 3A) and that of the proglobu-lar embryo is still uneven (Fig. 3B). The proglobular embryo; however, is isodiametric and globular (Fig. 3C). The cell clusters and proglobu-lar embryos appeared to modify their irreguproglobu-lar surfaces by division and elongation of their su-perficial cells to yield the smooth surface of em-bryo rounded in shape, and it is likely that the superficial cells of the structure are important in
Fig. 2. Formation of a globular embryo from part of a cell cluster. A cell cluster formed 7 days after initiation of culture in 2,4-D-free medium was transferred to fresh 2,4-D-free medium. An arrowhead (1 day of culture) indicates part of the cell cluster that separated from the rest and independently generated a globular embryo. Numbers refer to numbers of days after the start of culture in fresh 2,4-D-free medium. Bar=50 mm for all images.
Fig. 1. Serial observations of transformation of two selected single cells (A and B) to cell clusters. Regenerated plantlets were cultured in MS liquid medium for 24 h, washed thor-oughly with free medium, and then cultured in 2,4-D-free medium. After culture for 3 days, single cells were collected from a heterogeneous population of cells on nylon screens with 84 and 41mm pores and was cultured in
condi-tioned medium as described in Section 2. Numbers indicate the numbers of days after the single cell had been transferred to the conditioned medium. Bar=50mm for all images.
devel-opment of the heart-shaped embryo. We found three such cases of asynchronous formation of cotyledons among 36 heart-shaped embryos, which were formed in this serial observation. A heart-shaped embryo, characterized by a bilater-ally symmetrical structure with two cotyledons was initiated directly from a cell cluster, without a typical globular stage (Fig. 6). Thus, a half-moon-shaped cell cluster (2-day-old culture in Fig. 6) that had developed from a starting structure un-derwent active cell division on its flat side to
Fig. 4. Typical embryogenesis from a cell cluster to a heart-shaped embryo via a globular embryo. The cell cluster was prepared and cultured as described in the text. Numbers indicate the number of days after transfer. Bar=50mm for all
images.
Fig. 3. SEM images from cell cluster to heart-shaped em-bryos. A, cell cluster; B, proglobular embryo; C, globular embryos; D, proglobular embryo; E, globular embryo; F, heart-shaped embryo; G, appearance of cotyledonary primor-dia at the apex of a globular embryo.
Fig. 3G shows a cotyledonary primordium pro-truding from the apical pole of a globular embryo. Cell division has occurred predominantly in the cotyledonary region and the cells in the cleft of the cotyledons have ceased to divide, generating a heart-shaped embryo. The protruding cells of the cotyledonary lobe are arranged around the central pole by cell division and cell elongation.
The superficial cells of proglobular embryos were of various sizes and randomly arranged on the surface (Fig. 3D). These larger superficial cells probably developed to yield a globular embryo by repeated cycles of random elongation and division. At the late globular stage (Fig. 3E), cell division
Fig. 6. Embryogenesis from a cell cluster to heart-shaped embryo via an atypical pathway. Numbers indicate the num-ber of days after transfer. Bar=50 mm.
Fig. 5. Globular embryo with cotyledonary primordia. Num-bers indicate the number of days after transfer. Bar=50 mm
for all images.
occurred parallel to the axis of the embryo. Such late globular embryos seemed to correspond to the oblong embryos described previously by Schi-avone and Cooke [12]. It is likely that both apical poles become origins for the outgrowth of the two cotyledons and the beginning of the development of the radicle. In the early heart-shaped embryo (Fig. 3F), which is characterized by the outgrowth of the two cotyledons, the superficial cells of the cotyledonary lobe and larger cells, which still re-mained in the central part of the surface of the embryo, divide and then elongate parallel to the axis. The orientation of the division and elonga-tion of superficial cells is likely to be a determinant of morphogenesis from the globular stage to heart-shaped stage.
shapes and uneven surfaces. SEM studies by Xu and Bewley [3] revealed the rough surface of so-matic embryo in alfalfa and showed them to be different from smooth zygotic embryos.
The polarity of development of the heart-shaped embryo from the globular embryo is regulated by the polar transport of auxin, which is intimately involved in the initiation and maintenance of po-larized growth in developing embryos [14,15]. DNA synthesis occurs actively in localized regions of cell clusters immediately before the develop-ment to globular embryos [16]. Thus, it is likely that the unusual sequence of morphological events, by which cells as part of a cell cluster or embryo divided actively to initiate unusual cotyle-donary primordia in somatic embryogenesis (Fig. 6), was caused by locally elevated levels of auxin and abnormal polar transport of auxin within the cell cluster and embryo. In addition, differences in sensitivity to localized endogenous auxin might be involved in the transition from a cell cluster with an irregular shape to a globular embryo with an isodiametric and spherical shape.
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