Plant Cell Reports
Plant Cell Reports (1995) 15:242-247
~ Springer-Verlag1995
Developmental patterns during direct somatic embryogenesis in protoplast cultures of european larch (Larix decidua Mill.) Jonas Korlach and Kurt Zoglauer Humboldt-Universit/it zu Berlin, Mathematisch-NaturwissenschafllicheFakultgt I, Institut ffir Biologie, Invalidenstr. 43, D-10115 Berlin, Germany Received 6 January 1995/Revised version received 15 March 1995- Communicated by H. Lbrz
Summary. Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 gM both 2,4-dichlorophenoxyacetie acid or indole-3-acetie acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonai mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenie program.
Key words: Auxin - Larix decidua Mill. - Protoplasts Single cell monitoring - Somatic embryogenesis Abbreviations: BAP: N~-benzylaminopurine; 2,4-D: 2,4-dichlorophenoxyacetic acid; IAA: indole-3-acetic acid; MES: 2-(N-morpholino)ethanesulfonic acid; PEM: proembryonal mass
Introduction Somatic embryogenesis provides an ideal experimental system for investigations on cell differentiation as well as totipotency expression in plants (Thorpe 1988). Although biochemical, cytological and physiological studies of in vitro conifer embryogenesis have resulted in improved knowledge about the origin of somatic embryos, morphogenetie phenomena and comparability to Correspondence to: K. Zoglauer
zygotic embryos, our understanding of the stimuli and conditions necessary for the control of these processes is still minimal (Tautorns et al. 1991). Particularly, ceU division patterns preceding meristem formation remain undescribed (yon Aderkas ct al. 199 I). In the genus Larix, cytological descriptions of early division and differentiation patterns of morphogenetic protoplasts are available for L. x eurolepis and haploid or diploid L. decidua Mill. (Klimaszcwska 1989a; yon Aderkas 1992; Zoglauer et al. 1992) as well as for somatic embryos obtained from megagametophytes of two Larix species (Nagmani and Bonga 1985; yon Aderkas and Bonga 1988; yon Aderkas et al. 1990). Structural comparisons between somatic and natural zygotic embryogenesis have been carried out by yon Aderkas et al. (1991). However, this study was focussed on somatic embryo development from the megagametophytic explant so that systematic observations of early stages during direct somatic embryogenesis which starts from an isolated cell initial are still missing. In addition, one major problem in respect to the comparability of these two processes as well as investigations on endogenous factors during polarity formation consists in the general necessity of auxins and cytokinins in gymnosperm culture media for somatic embryo initiation, maintenance of embryogenic suspension cultures and regeneration from protoplasts (Klimaszewska 1989b; Bellarosa et al. 1992; Attree et al. 1987). In the present study, a detailed characterization of early developmental stages is given for protoplast cultures of European larch (Larix decidua Mill.) through single cell monitoring, and compared to naturally occurring zygotic embryogenesis. In addition, the differentiated responses of the cells to various concentrations of IAA, 2,4-D and BAP as well as the fates of coenocytic cells which originated from spontaneous fusion during the protoplast isolation process (Behrendt et al. 1994) are also described.
243 Material and methods Donor material The embryogenic culture used for protoplast experiments has been induced in our lab from an immature zygotic embryo in 1990. This suspension (Fig. 2) is propagated weekly according to Krogstrup (1990) by subculture in a modified liquid MSG medium (Beewar et al. 1990) supplemented with 5ktM 2,4-D (Serva), 2 IxM BAP (Sigma), and 2 ~tM kinetin ($erva) (Klimaszewska 1989b). 5 or 10 ml of sedimented cell volume are transferred into 250 or 500 ml Erlenmeyer flasks with 50 or 100 ml of fresh medium, respectively. They are kept in the dark at 22+1~ on a rotating shaker at 70 rpm. Preparation o f matrix support meshes. For single cell monitoring, a method previously described by Golds et al. (1992) was modified. In order to generate thin films of calcium alginate, squares of a nylon mesh (1 x I mm mesh size, 0.4 mm thick) were cut to fit exactly inside 60 mm Petri dishes (ensuring no free movement within the dish). 5 grid strips of both horizontal and vertical directions were pulled out at regular distances with a tweezers - the resulting 25 squares (2 x 2 nun) were used for observations of developmental patterns of embedded protoplasts (Fig. 1). The meshes were autoclaved and placed under steril conditions onto a solidified medium (Difco agar 1%) containing 0.4 M sorbitol and 20 mM CaCl 2 x 2 H 2 0 in 60 mm Petri dishes.
The suspension was mixed with an equal volume of 2 % (w/v) alginie acid (sodium salt, medium viscosity, Sigma) in 0.5 M sorbitol to a final protoplast density ot"2.5 x 104 and plated in aliquots of 0.6 red onto the Ca-agar dishes (with the prepared mesh) where protoplasts were immobilized by ionotropie gelation of the calcinm-alginate layer within 1 h. The meshes with the calcium-alginate films were then transferred into 60 mm Pelri dishes containing 5 ml of modified DCR (Gupta and Durzan 1985; Zoglaner et al. 1992) or PCM4 medium (Attree et al. 1989), respectively, with a phytohormone content of 5 pM BAP and 10 ~tM 2,4-D. For experiments with variations in the hormone eompositien (Table 1), the respective amounts of BAP, 2,4-1) or IAA (Serva) were added (as 0 . 0 1 % (w/v) sterile solutions) to a hormone-free solution of culture medium. The cultures were kept in the dark at 22+1 ~ Table 1. Phytohormone concentrations [p-M] in the controls (1-3) and
experiments varying the growth regulator strength (4-8) Experiment BAP 2,4-D or IAA
1 0 0
2 5 0
3 0 10
4 1.25 2.5
5 2.5 5
6 3.75 7.5
7 5 10
8 10 20
Results
Fig. 1. Prepared mesh in which protoplasts were embedded for single cell trackinD Bat = 1 en~ Protoplast isolation and culture. 5 days from subculture, rapidly proliferating embryogenio suspension material was used for protoplast isolation which has been described previously (Behrendt 1994). Briefly, the culture medium was replaced by an osmotic-stabilizer-solution consisting of 0.5 M sorbitol, 3 mM CaCI 2 x 2 H20, and 5 mM MES (Serva) at pH 5.8 (yielding ca. 550 mOsm/kg) to which the enzyme mixture containing (as final concentrations) 0.125 % (w/v) Ceilulase TC, 0.25 % (w/v) Pectinase (Serva), and 0.0125 % (w/v) Driselase (Sigma) was added for gentle digestion. In order to induce spontaneous protoplast fusion, the concentrations of all enzymes were doubled. The 100 mm Petri dishes were incubated for 15 h at 22 ~ in the dark on a rocking shaker at 10 rpm. The resulting protoplast suspension was passed through a 63 tun steel sieve and sedimented by centrifugation for 10 rain at 100 x g, 24 % (w/v) FicoU-400 solution (Sigma) in the osmotic-stabilizersolution (FS) was added to the pellet and some supematant to a final Ficoll concentration of 4 %. The suspension was carefully overlayed with 1.5 ml of washing solution (Schlangstedt et al. 1994). After 10 rain centrifugation at 100 x g the suspensor protoplasts floated on top of the density border layer whereas embryo protoplasts or fusion products were found in the sediment. Collection and cleaning of the latter was achieved by resuspension in 24 % FS and overlaying with 1 ml 0.5 M sorbitol. After 25 rain centrifugation at 100 x g this protoplast fraction was removed from the interface, diluted in 0.5 M sorbitol and counted.
Prior to embryo differentiation, a proembryonal mass (PEM) developed. The process of somatic embryogenesis from a single isolated protoplast (Fig. 3A) to an embryo consisting of apically situated, cytoplasmicaUy dense cells and a secondary suspensor composed of embryonal tube cells (Fig. 3G) was completed after approximately four weeks. Plating efficiencies (determined after 14 days of culture) ranged between 7 and 19 %. First cell divisions not exhibiting any morphological polaxity occurred after two days. Additional symmetrical cell divisions lead to the formation of embryogenic colonies, often containing a number of coenocytic cells (Fig. 3B, arrows). By analogy with Becwar et al. (1988), this structure will be referred to as the proembryonal mass (PEM) since it is composed of an unorganized cell aggregate lacking any ordered secondary suspensor. It is here preferred to the term proembryogenic mass commonly used in angiosperm embryology (Halperin 1966) for the reason that this structure possesses postinductive embryogenic capacity and thus represents a distinctive developmental stage during the ongoing process of somatic embryo regeneration. On the other hand, it has to be separated from the proembryo described by Schopf (1943) since it does not exhibit any of the therein described conservative cell arrangement patterns. Vacuolation and subsequent degeneration of individual cells in the PEA# is caused by supplemented auxin. Depending on the concentrations of growth regulators, different phenomena were observed in DCR culture medium. A representative sequence of successive stages in the embryo development from protoplasts cultured in DCR containing an auxin (Exp. 4-8) is shown in Fig. 3A-G. After 3-4 symmetrical cell divisions a loose cluster of ca. 15 cells was formed (Fig. 3C). 14-17 days after
244
245
Fig. 2. A: Larix decidua Mill. embryogenic suspension culture used for protoplast isolation. Bar = 200/am. B: Typical somatic embryo with suspensor. Bar = 50/am. Fig. 3. Time-lapse series of somatic embryogenesis from a Larix decidua Mill. protoplast in DCR supplemented with 5/aM BAP and 10 ~tM IAA. A: Protoplast in the alginate matrix 2 days after isolation. Note the large nucleoli and a low cytoplasm-nucleus ratio. B: Day 11. Four-celled stage. Note the binucleated cell (arrows). C: Day 15. Further symmetrical cell divisions lead to a loose cell cluster (PEM) in which the first vacuolafion of an individual cell occurs (arrow). D: Day 19. Three different regions are recognizable in the PEM: elongated suspensor-like cells (large arrows), degenerated cells adjacent to them (arrow heads), and a proliferating region consisting of small and cytoplasmically dense cells (arrows). E: Day 25. The suspensor-like cells degenerate (large arrow), and various compact cell clusters arise (arrows). F: Day 31. The newly formed suspensor cells (arrow heads) push the embryos (arrows) from the PEM. Note the occurrence of cleavage polyembryony. G: Day 34. Section from the embryonal mass showing a protoplast-defived embryo with a distinct secondary suspensor. A: Bar = 20 ~tm. B-E,G: Bar = 50 ~tm. F: Bar = 100 lain.
Fig. 4. Patterns of embryo regeneration in phytohormone-free DCR. A: After 20 days of culture, a compact cluster ofembryogenic cells has been formed. B: Day 25. Parallel suspensor cells (large arrows) separate the embryos (arrows) from the later on degenerating cells at the distal end (arrow heads). C: Protoplast-derived embryo with a well-developed secondary suspensor after 34 days. A,B: Bar = 50 ~tm. C: Bar = 100 ~tm.
Fig. 5. In PCM4 culture medium, protoplasts only proliferate to PEM's without signs of differentiation into embryos. Already 2,5/aM IAA cause a vacuolation effect on peripheral cells (arrows). Bar = 100 ~tm.
Fig. 6. Development ofoligonucleated protoplasts ofLarix decidua Mill. A: Tetranucleated protoplast one day a~er isolation. Bar = 20 ~tm.B: The syncytium has cellularized into four individual cells after seven days, and performs a normal embryogenic program. Bar = 50 ~tm.
246 protoplast isolation, one or more individual cells died or became very large and highly vacuolated before subsequent degeneration (Fig. 3D). Embryonal cells adjacent to these polymorphous suspensor-like calls often degenerated as well, resulting in an area of dead cells within the PEM. This simultaneous proliferation and decease without any incidences of morphogenetic differentiation continued for about three weeks. Finally, very compact dusters of cytoplasmically dense cells appeared, usually at the periphery of the PEM (Fig. 3E). These clusters representing embryos were subsequently pushed away from the PEM within a short number of days through the rapid elongation of the newly formed, more or less parallel oriented embryonal tube cells forming the suspensor (Fig 3F, arrow heads). With increasing auxin concentrations, developmental patterns shifted from morphogenesis toward a dominance of the proliferating phase. At high auxin concentrations (Exp. 3, 7 & 8), differentiation into distinct embryogenic structures very rarely occurred, and not before six weeks after isolation. However, since more and more snspensor-like cells appeared in the PEM, the effective growth rate was even slower than in cultures with a lower auxin content (Exp. 4 & 5). In the latter, the first immature embryos were visible after four weeks. In culture medium lacking any phytohormones, direct embryogenesis is faster and better coordinated Embryogenic competence was not at all diminished in auxin-free DCR medium (EXP. 1 & 2). Instead, protoplasts usually developed into a single embryo within three weeks (Fig. 4C). In these experiments, no elongation and vacuolation of protoplast-derived cells was observed prior to the formation of a snspensor. Thus, the PEM consisted of a very compact aggregate of small and cytoplasmicaUy dense, always mononucleated cells, alreadyreminiscentof the dome-shaped structure of an embryo (Fig. 4A). After approximately two weeks, a few cells on one side of the PEM became highly vacuolated. With their degeneration, a typical secondary suspensor developed adjacently, pushing the embryo away from its initial position in the alginate matrix. Delayed cleavage polyembryony was frequently observed in these experiments (Fig. 4B, arrows). Cells at the distal end of the embryo degenerated further, but new embryonal tube cells were constantly generated so that the snspensor sometimes obtained a considerable length (Fig. 4C). Generally, variations in the BAP content did not result in remarkable alterations from the described developmental patterns, but they were not investigated in detail in the absence of auxin. Distinct differences in the extent of embryogenesis were observed in respect to the used culture medium. Whereas somatic embryos typical to those in the source tissue culture (Fig. 213) developed in DC1L protoplasts growing in PCM4 only proceeded to proliferate, thus forming loose PEM's without any formation of organized embryonal structures (Fig. 5).
Except for auxin-free variants, large suspensor-like cells also appeared in PCM4 cultures (Fig. 5, arrows). In connection with an increase in the number of those cells formed per PEM, the growth rate was drastically reduced with higher auxin concentrations. At concentrations of 10 ~tM auxin and higher (Exp. 3, 7 & 8), protoplasts usually were not able to perform more than four cell division cycles. Oligonucleated protoplasts were capable to cellularize without prior karyokinesis, and developed into embryos. Although the majority of fusion products soon deceased, some coenocytic protoplasts (Fig. 6A) rapidly cellularized into a loosely packed cell aggregate (Fig. 6B), and showed a morphogenetic program indistinguishable from mononucleated cells. No karyokinesis was observed before cytoplasmic segmentation. Multinucleated protoplasts containing more than four nuclei, however, did not perform a complete cytokinesis into individual cells. Instead, long and deep furrows appeared on the surface of the syncytium before its degeneration.
Discussion
Somatic embryogenesis from protoplasts in an environment free of any exogenous plant growth regulators demonstrates the high amount of morphogenetic autonomy the initial cell exhibiting embryogenic competence possesses. In this environment, the development is comparable to naturally occurring zygotic embryogenesis since important types of cell differentiation are found. The asymmetric metabolic conditions necessary for the inception of polarized growth have to be derived from factors inherent in the spatial and temporal organizatiun of the PEM. This is the first report of a model system in conifers which allows further investigations on polarity formation and control mechanisms of developmental processes mediated by endogenous phytohormones without disturbances caused by these growth regulators in the culture medium. However, in vitro embryogeny lacks the precise quantitative aspects that are found in vivo. Particularly, early patterns of cell divisions are not identical to zygotic embryogenesis. Instead of an ordered formation of cell tiers followed by their subsequent differentiation (Schopf 1943), a globular aggregate of morphologically equal, small and cytoplasmically dense cells is formed. The isotropic supply of nutrients in the culture dish, connected with the lack of mechanical and physiological controlling factors provided by the archegonium and later on by corrosion cavity which leads to a diminished polarity of the tissue,is a possible explanation for this response. The frequent incidence of a form of polyembryony in which many independent embryos develop from one PEM supports this hypothesis. Small, but highly vacuolated cells formed later on at
247 one end of the PEM can be considered as being homologous to rosette cells described for zygotic embryogeny (Schopf 1943). The same is tree for embryonal tube cells in respect to the secondary snspensor. In vitro, they appear in a less tight connection to each other, but are otherwise very similar. The finding that all early cell divisions were symmetrical also in media containing an auxin is in contrast to observations made by Klimaszewska (1989a). However, since morphological identity does not imply physiological symmetry the cells probably remain polar until they can express this bipolarity in an appropriate environment. Exogenous auxin mediates a dominance of non-polar proliferation. Later stages in larch embryogeny only develop when the relative size of the PEM stipulates anisotropic conditions, thus permitting cell-cell-interactions which lead then to a directed morphogenetic activity. This is also well known for angiosperms (e.g. Goldberg et al. 1994) where, initially, only the removal of auxin results in embryogenesis from proembryogenic masses (e.g. Halperin 1966, Halperin and Jensen 1967). In gynmosperms, early developmental stages are more distinct through the appearance of a well-developed snspensor. Therefore, such sensitive in vitro cultures represent an excellent model system allowing selective influencing of embryogenic processes from the outside. Enlarged and vacuolated snspensor-like cells that start to occur very early in the process of somatic embryo regeneration from protoplasts have been described previously by other authors (Klimaszewska 1989a, Attree et al. 1987). Their formation can now be clearly attributed to supplemented auxin. They do not represent embryonal tube cells since they appear independently at various locations within the PEM. Additionally, a strong dependency of the number of those cells developing per PEM from the auxin concentration was found. Even 9a low concentration of auxin (2.5 BM) disturbs the morphogenetic process due to its well-known effect of prometing cell elongation, thus withdrawing embryogenic cells from a further development. The large size and highly irregular shape of the ceils is probably due to the excessive and isotropic supply of nutrients and phytohormones, connected with a diminution of correlative restrictions made by adjacent cells. It is possible that the occurrence of coenocytic cells during embryogenesis is a result of 2,4-D or IAA treatment as well since auxins are known to cause irregularities during cell growth in cultures of other organisms, such as Pisum (Gantchev and Bleiss 1994). The inability to perform a development towards immature embryos in PCM4 medium cannot have its origins in hormonal requirements. The crucial factor why protoplasts stop at the PEM stage of embryogeny is currently investigated in our lab. Cytoplasmic segmentation of oligonucleated products
of spontaneous protoplast fusions is of cytological interest since, contrary to findings of other authors (von Aderkas 1992), no preceding karyokinesis was observed. Furthermore, it is apparent that in contrast to free nuclei which are common during early zygotic embryogenesis the cytoskeleton became highly disturbed during the fusion process and has to create individual compartments anew (Stax6n et al. 1994). In rive, the cytoskeleton and nuclei arrangements always remain highly ordered. Comparative investigations on a possible creation of spindles between two nuclei as a prerequisite of cell wall formation and connected aspects might improve our knowledge on the ultrastmctural organization of the cytoplasm.
Acknowledgements. The authors wish to thank Dr. H. Dembny for the initiation and maintenance of the embryogenic suspension cultures. In addition, we would like to acknowledge the contributions of U. Behrendt regarding the development of this protoplast culture system in our lab.
References Attree SM, Bekkaoui F, Dunstan DI, Fowke LC (1987) Plant Cell Rep 6: 480-483 Attree SM, Dunstan DI, Fowke LC (1989) Can J Bet 67:1790-1795 Becwar MR, Warm SR, Johnson MA, Verhagen SA, Feirer RP, Nagmani R (1988) In: Ahuja MR (eds) Somatic Cell Genetics of Woody Plants. Kluwer Academic, Dordrecht, pp 1-18 Becwar MR, Nagmani R, Warm SR (1990) Can J For Res 20:810-817 Behrendt U (1994) Dissertation, Humboldt-Univ. Berlin: 11-12 Bebxendt U, Kodach J, Zoglaner K (1994) VIIIth IAPTC Congress Abstracts. Firenze, p 194 Bellarosa R, Me LH, yon Arnold S (1992) Ann Bet: 199-206 Gantchev L, Bleigs W (1994) Biol Plant 36(3): 335-342 Goldberg RB, Depaiva G, Yadegari R (1994) Science 266:605-614 Golds JG, Babczinsky J, Rauscher (3, Keep H-U (1992) J Plant Physiol 140:582-587 CJUptaPK, Durzan DJ (1985) Plant Cell Rep 4:177-179 Haiperin W (1966) Amer J Bet 53(3): 443-453 Halperin W, Jensen WA (1967) J Ultras(ruet Res 18:428-443 Klimaszewska K (1989a) Plant Cell Rep 8:440-444 Klimaszewska K (1989b) Plant Sci 63:95-103 Krogst~p P (1990) Plant Sci 72:115-123 Nagmani R, Bonga JM (1985) Can J For Rcs 15:1088-1091 Schlangstedt M, Zoglauer K, Lenzner S, Hermans B, Jacobs M (1994) J Plant Physiol 143:227-233 Schopf JM (1943) The embryology of Larix. Illinois Biological Monographs 19:1-97 Stax&l I, Klimaszewska K, Bomman CH (1994) Physiol Plant 91: 680-686 Tantoms TE, Fowke LC, Dunstan DI (1991) Can J Bot 69:1873-1899 Thorpe TA (1988) ISI Atlas Sci Anita Plant Sci 1:81-88 van Aderkas P, Bonga JM (1988) Amer J Bet 75(5): 690-700 yon Aderkas P, Klimaszewska K, Bonga JM (1990) Can J For Res 20: 9-14 yon Aderkas P, Bonga J, Klimaszewska K, Owens J (1991) In: Ahuja MR (eds) Woody Plant Biotechnology. Plenum Press, New York, pp 139-155 von Aderkas P (1992) Can J For Res 22:397-402 Zoglauer K, Dembny H, Behrendt U (1992) Wigs Zeitschrifl HumboldtUniv Berlin, R Mat/Nat 41(3): 51-62
Plant Cell Reports (1995) 15:242-247
~ Springer-Verlag1995
Developmental patterns during direct somatic embryogenesis in protoplast cultures of european larch (Larix decidua Mill.) Jonas Korlach and Kurt Zoglauer Humboldt-Universit/it zu Berlin, Mathematisch-NaturwissenschafllicheFakultgt I, Institut ffir Biologie, Invalidenstr. 43, D-10115 Berlin, Germany Received 6 January 1995/Revised version received 15 March 1995- Communicated by H. Lbrz
Summary. Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 gM both 2,4-dichlorophenoxyacetie acid or indole-3-acetie acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonai mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenie program.
Key words: Auxin - Larix decidua Mill. - Protoplasts Single cell monitoring - Somatic embryogenesis Abbreviations: BAP: N~-benzylaminopurine; 2,4-D: 2,4-dichlorophenoxyacetic acid; IAA: indole-3-acetic acid; MES: 2-(N-morpholino)ethanesulfonic acid; PEM: proembryonal mass
Introduction Somatic embryogenesis provides an ideal experimental system for investigations on cell differentiation as well as totipotency expression in plants (Thorpe 1988). Although biochemical, cytological and physiological studies of in vitro conifer embryogenesis have resulted in improved knowledge about the origin of somatic embryos, morphogenetie phenomena and comparability to Correspondence to: K. Zoglauer
zygotic embryos, our understanding of the stimuli and conditions necessary for the control of these processes is still minimal (Tautorns et al. 1991). Particularly, ceU division patterns preceding meristem formation remain undescribed (yon Aderkas ct al. 199 I). In the genus Larix, cytological descriptions of early division and differentiation patterns of morphogenetic protoplasts are available for L. x eurolepis and haploid or diploid L. decidua Mill. (Klimaszcwska 1989a; yon Aderkas 1992; Zoglauer et al. 1992) as well as for somatic embryos obtained from megagametophytes of two Larix species (Nagmani and Bonga 1985; yon Aderkas and Bonga 1988; yon Aderkas et al. 1990). Structural comparisons between somatic and natural zygotic embryogenesis have been carried out by yon Aderkas et al. (1991). However, this study was focussed on somatic embryo development from the megagametophytic explant so that systematic observations of early stages during direct somatic embryogenesis which starts from an isolated cell initial are still missing. In addition, one major problem in respect to the comparability of these two processes as well as investigations on endogenous factors during polarity formation consists in the general necessity of auxins and cytokinins in gymnosperm culture media for somatic embryo initiation, maintenance of embryogenic suspension cultures and regeneration from protoplasts (Klimaszewska 1989b; Bellarosa et al. 1992; Attree et al. 1987). In the present study, a detailed characterization of early developmental stages is given for protoplast cultures of European larch (Larix decidua Mill.) through single cell monitoring, and compared to naturally occurring zygotic embryogenesis. In addition, the differentiated responses of the cells to various concentrations of IAA, 2,4-D and BAP as well as the fates of coenocytic cells which originated from spontaneous fusion during the protoplast isolation process (Behrendt et al. 1994) are also described.
243 Material and methods Donor material The embryogenic culture used for protoplast experiments has been induced in our lab from an immature zygotic embryo in 1990. This suspension (Fig. 2) is propagated weekly according to Krogstrup (1990) by subculture in a modified liquid MSG medium (Beewar et al. 1990) supplemented with 5ktM 2,4-D (Serva), 2 IxM BAP (Sigma), and 2 ~tM kinetin ($erva) (Klimaszewska 1989b). 5 or 10 ml of sedimented cell volume are transferred into 250 or 500 ml Erlenmeyer flasks with 50 or 100 ml of fresh medium, respectively. They are kept in the dark at 22+1~ on a rotating shaker at 70 rpm. Preparation o f matrix support meshes. For single cell monitoring, a method previously described by Golds et al. (1992) was modified. In order to generate thin films of calcium alginate, squares of a nylon mesh (1 x I mm mesh size, 0.4 mm thick) were cut to fit exactly inside 60 mm Petri dishes (ensuring no free movement within the dish). 5 grid strips of both horizontal and vertical directions were pulled out at regular distances with a tweezers - the resulting 25 squares (2 x 2 nun) were used for observations of developmental patterns of embedded protoplasts (Fig. 1). The meshes were autoclaved and placed under steril conditions onto a solidified medium (Difco agar 1%) containing 0.4 M sorbitol and 20 mM CaCl 2 x 2 H 2 0 in 60 mm Petri dishes.
The suspension was mixed with an equal volume of 2 % (w/v) alginie acid (sodium salt, medium viscosity, Sigma) in 0.5 M sorbitol to a final protoplast density ot"2.5 x 104 and plated in aliquots of 0.6 red onto the Ca-agar dishes (with the prepared mesh) where protoplasts were immobilized by ionotropie gelation of the calcinm-alginate layer within 1 h. The meshes with the calcium-alginate films were then transferred into 60 mm Pelri dishes containing 5 ml of modified DCR (Gupta and Durzan 1985; Zoglaner et al. 1992) or PCM4 medium (Attree et al. 1989), respectively, with a phytohormone content of 5 pM BAP and 10 ~tM 2,4-D. For experiments with variations in the hormone eompositien (Table 1), the respective amounts of BAP, 2,4-1) or IAA (Serva) were added (as 0 . 0 1 % (w/v) sterile solutions) to a hormone-free solution of culture medium. The cultures were kept in the dark at 22+1 ~ Table 1. Phytohormone concentrations [p-M] in the controls (1-3) and
experiments varying the growth regulator strength (4-8) Experiment BAP 2,4-D or IAA
1 0 0
2 5 0
3 0 10
4 1.25 2.5
5 2.5 5
6 3.75 7.5
7 5 10
8 10 20
Results
Fig. 1. Prepared mesh in which protoplasts were embedded for single cell trackinD Bat = 1 en~ Protoplast isolation and culture. 5 days from subculture, rapidly proliferating embryogenio suspension material was used for protoplast isolation which has been described previously (Behrendt 1994). Briefly, the culture medium was replaced by an osmotic-stabilizer-solution consisting of 0.5 M sorbitol, 3 mM CaCI 2 x 2 H20, and 5 mM MES (Serva) at pH 5.8 (yielding ca. 550 mOsm/kg) to which the enzyme mixture containing (as final concentrations) 0.125 % (w/v) Ceilulase TC, 0.25 % (w/v) Pectinase (Serva), and 0.0125 % (w/v) Driselase (Sigma) was added for gentle digestion. In order to induce spontaneous protoplast fusion, the concentrations of all enzymes were doubled. The 100 mm Petri dishes were incubated for 15 h at 22 ~ in the dark on a rocking shaker at 10 rpm. The resulting protoplast suspension was passed through a 63 tun steel sieve and sedimented by centrifugation for 10 rain at 100 x g, 24 % (w/v) FicoU-400 solution (Sigma) in the osmotic-stabilizersolution (FS) was added to the pellet and some supematant to a final Ficoll concentration of 4 %. The suspension was carefully overlayed with 1.5 ml of washing solution (Schlangstedt et al. 1994). After 10 rain centrifugation at 100 x g the suspensor protoplasts floated on top of the density border layer whereas embryo protoplasts or fusion products were found in the sediment. Collection and cleaning of the latter was achieved by resuspension in 24 % FS and overlaying with 1 ml 0.5 M sorbitol. After 25 rain centrifugation at 100 x g this protoplast fraction was removed from the interface, diluted in 0.5 M sorbitol and counted.
Prior to embryo differentiation, a proembryonal mass (PEM) developed. The process of somatic embryogenesis from a single isolated protoplast (Fig. 3A) to an embryo consisting of apically situated, cytoplasmicaUy dense cells and a secondary suspensor composed of embryonal tube cells (Fig. 3G) was completed after approximately four weeks. Plating efficiencies (determined after 14 days of culture) ranged between 7 and 19 %. First cell divisions not exhibiting any morphological polaxity occurred after two days. Additional symmetrical cell divisions lead to the formation of embryogenic colonies, often containing a number of coenocytic cells (Fig. 3B, arrows). By analogy with Becwar et al. (1988), this structure will be referred to as the proembryonal mass (PEM) since it is composed of an unorganized cell aggregate lacking any ordered secondary suspensor. It is here preferred to the term proembryogenic mass commonly used in angiosperm embryology (Halperin 1966) for the reason that this structure possesses postinductive embryogenic capacity and thus represents a distinctive developmental stage during the ongoing process of somatic embryo regeneration. On the other hand, it has to be separated from the proembryo described by Schopf (1943) since it does not exhibit any of the therein described conservative cell arrangement patterns. Vacuolation and subsequent degeneration of individual cells in the PEA# is caused by supplemented auxin. Depending on the concentrations of growth regulators, different phenomena were observed in DCR culture medium. A representative sequence of successive stages in the embryo development from protoplasts cultured in DCR containing an auxin (Exp. 4-8) is shown in Fig. 3A-G. After 3-4 symmetrical cell divisions a loose cluster of ca. 15 cells was formed (Fig. 3C). 14-17 days after
244
245
Fig. 2. A: Larix decidua Mill. embryogenic suspension culture used for protoplast isolation. Bar = 200/am. B: Typical somatic embryo with suspensor. Bar = 50/am. Fig. 3. Time-lapse series of somatic embryogenesis from a Larix decidua Mill. protoplast in DCR supplemented with 5/aM BAP and 10 ~tM IAA. A: Protoplast in the alginate matrix 2 days after isolation. Note the large nucleoli and a low cytoplasm-nucleus ratio. B: Day 11. Four-celled stage. Note the binucleated cell (arrows). C: Day 15. Further symmetrical cell divisions lead to a loose cell cluster (PEM) in which the first vacuolafion of an individual cell occurs (arrow). D: Day 19. Three different regions are recognizable in the PEM: elongated suspensor-like cells (large arrows), degenerated cells adjacent to them (arrow heads), and a proliferating region consisting of small and cytoplasmically dense cells (arrows). E: Day 25. The suspensor-like cells degenerate (large arrow), and various compact cell clusters arise (arrows). F: Day 31. The newly formed suspensor cells (arrow heads) push the embryos (arrows) from the PEM. Note the occurrence of cleavage polyembryony. G: Day 34. Section from the embryonal mass showing a protoplast-defived embryo with a distinct secondary suspensor. A: Bar = 20 ~tm. B-E,G: Bar = 50 ~tm. F: Bar = 100 lain.
Fig. 4. Patterns of embryo regeneration in phytohormone-free DCR. A: After 20 days of culture, a compact cluster ofembryogenic cells has been formed. B: Day 25. Parallel suspensor cells (large arrows) separate the embryos (arrows) from the later on degenerating cells at the distal end (arrow heads). C: Protoplast-derived embryo with a well-developed secondary suspensor after 34 days. A,B: Bar = 50 ~tm. C: Bar = 100 ~tm.
Fig. 5. In PCM4 culture medium, protoplasts only proliferate to PEM's without signs of differentiation into embryos. Already 2,5/aM IAA cause a vacuolation effect on peripheral cells (arrows). Bar = 100 ~tm.
Fig. 6. Development ofoligonucleated protoplasts ofLarix decidua Mill. A: Tetranucleated protoplast one day a~er isolation. Bar = 20 ~tm.B: The syncytium has cellularized into four individual cells after seven days, and performs a normal embryogenic program. Bar = 50 ~tm.
246 protoplast isolation, one or more individual cells died or became very large and highly vacuolated before subsequent degeneration (Fig. 3D). Embryonal cells adjacent to these polymorphous suspensor-like calls often degenerated as well, resulting in an area of dead cells within the PEM. This simultaneous proliferation and decease without any incidences of morphogenetic differentiation continued for about three weeks. Finally, very compact dusters of cytoplasmically dense cells appeared, usually at the periphery of the PEM (Fig. 3E). These clusters representing embryos were subsequently pushed away from the PEM within a short number of days through the rapid elongation of the newly formed, more or less parallel oriented embryonal tube cells forming the suspensor (Fig 3F, arrow heads). With increasing auxin concentrations, developmental patterns shifted from morphogenesis toward a dominance of the proliferating phase. At high auxin concentrations (Exp. 3, 7 & 8), differentiation into distinct embryogenic structures very rarely occurred, and not before six weeks after isolation. However, since more and more snspensor-like cells appeared in the PEM, the effective growth rate was even slower than in cultures with a lower auxin content (Exp. 4 & 5). In the latter, the first immature embryos were visible after four weeks. In culture medium lacking any phytohormones, direct embryogenesis is faster and better coordinated Embryogenic competence was not at all diminished in auxin-free DCR medium (EXP. 1 & 2). Instead, protoplasts usually developed into a single embryo within three weeks (Fig. 4C). In these experiments, no elongation and vacuolation of protoplast-derived cells was observed prior to the formation of a snspensor. Thus, the PEM consisted of a very compact aggregate of small and cytoplasmicaUy dense, always mononucleated cells, alreadyreminiscentof the dome-shaped structure of an embryo (Fig. 4A). After approximately two weeks, a few cells on one side of the PEM became highly vacuolated. With their degeneration, a typical secondary suspensor developed adjacently, pushing the embryo away from its initial position in the alginate matrix. Delayed cleavage polyembryony was frequently observed in these experiments (Fig. 4B, arrows). Cells at the distal end of the embryo degenerated further, but new embryonal tube cells were constantly generated so that the snspensor sometimes obtained a considerable length (Fig. 4C). Generally, variations in the BAP content did not result in remarkable alterations from the described developmental patterns, but they were not investigated in detail in the absence of auxin. Distinct differences in the extent of embryogenesis were observed in respect to the used culture medium. Whereas somatic embryos typical to those in the source tissue culture (Fig. 213) developed in DC1L protoplasts growing in PCM4 only proceeded to proliferate, thus forming loose PEM's without any formation of organized embryonal structures (Fig. 5).
Except for auxin-free variants, large suspensor-like cells also appeared in PCM4 cultures (Fig. 5, arrows). In connection with an increase in the number of those cells formed per PEM, the growth rate was drastically reduced with higher auxin concentrations. At concentrations of 10 ~tM auxin and higher (Exp. 3, 7 & 8), protoplasts usually were not able to perform more than four cell division cycles. Oligonucleated protoplasts were capable to cellularize without prior karyokinesis, and developed into embryos. Although the majority of fusion products soon deceased, some coenocytic protoplasts (Fig. 6A) rapidly cellularized into a loosely packed cell aggregate (Fig. 6B), and showed a morphogenetic program indistinguishable from mononucleated cells. No karyokinesis was observed before cytoplasmic segmentation. Multinucleated protoplasts containing more than four nuclei, however, did not perform a complete cytokinesis into individual cells. Instead, long and deep furrows appeared on the surface of the syncytium before its degeneration.
Discussion
Somatic embryogenesis from protoplasts in an environment free of any exogenous plant growth regulators demonstrates the high amount of morphogenetic autonomy the initial cell exhibiting embryogenic competence possesses. In this environment, the development is comparable to naturally occurring zygotic embryogenesis since important types of cell differentiation are found. The asymmetric metabolic conditions necessary for the inception of polarized growth have to be derived from factors inherent in the spatial and temporal organizatiun of the PEM. This is the first report of a model system in conifers which allows further investigations on polarity formation and control mechanisms of developmental processes mediated by endogenous phytohormones without disturbances caused by these growth regulators in the culture medium. However, in vitro embryogeny lacks the precise quantitative aspects that are found in vivo. Particularly, early patterns of cell divisions are not identical to zygotic embryogenesis. Instead of an ordered formation of cell tiers followed by their subsequent differentiation (Schopf 1943), a globular aggregate of morphologically equal, small and cytoplasmically dense cells is formed. The isotropic supply of nutrients in the culture dish, connected with the lack of mechanical and physiological controlling factors provided by the archegonium and later on by corrosion cavity which leads to a diminished polarity of the tissue,is a possible explanation for this response. The frequent incidence of a form of polyembryony in which many independent embryos develop from one PEM supports this hypothesis. Small, but highly vacuolated cells formed later on at
247 one end of the PEM can be considered as being homologous to rosette cells described for zygotic embryogeny (Schopf 1943). The same is tree for embryonal tube cells in respect to the secondary snspensor. In vitro, they appear in a less tight connection to each other, but are otherwise very similar. The finding that all early cell divisions were symmetrical also in media containing an auxin is in contrast to observations made by Klimaszewska (1989a). However, since morphological identity does not imply physiological symmetry the cells probably remain polar until they can express this bipolarity in an appropriate environment. Exogenous auxin mediates a dominance of non-polar proliferation. Later stages in larch embryogeny only develop when the relative size of the PEM stipulates anisotropic conditions, thus permitting cell-cell-interactions which lead then to a directed morphogenetic activity. This is also well known for angiosperms (e.g. Goldberg et al. 1994) where, initially, only the removal of auxin results in embryogenesis from proembryogenic masses (e.g. Halperin 1966, Halperin and Jensen 1967). In gynmosperms, early developmental stages are more distinct through the appearance of a well-developed snspensor. Therefore, such sensitive in vitro cultures represent an excellent model system allowing selective influencing of embryogenic processes from the outside. Enlarged and vacuolated snspensor-like cells that start to occur very early in the process of somatic embryo regeneration from protoplasts have been described previously by other authors (Klimaszewska 1989a, Attree et al. 1987). Their formation can now be clearly attributed to supplemented auxin. They do not represent embryonal tube cells since they appear independently at various locations within the PEM. Additionally, a strong dependency of the number of those cells developing per PEM from the auxin concentration was found. Even 9a low concentration of auxin (2.5 BM) disturbs the morphogenetic process due to its well-known effect of prometing cell elongation, thus withdrawing embryogenic cells from a further development. The large size and highly irregular shape of the ceils is probably due to the excessive and isotropic supply of nutrients and phytohormones, connected with a diminution of correlative restrictions made by adjacent cells. It is possible that the occurrence of coenocytic cells during embryogenesis is a result of 2,4-D or IAA treatment as well since auxins are known to cause irregularities during cell growth in cultures of other organisms, such as Pisum (Gantchev and Bleiss 1994). The inability to perform a development towards immature embryos in PCM4 medium cannot have its origins in hormonal requirements. The crucial factor why protoplasts stop at the PEM stage of embryogeny is currently investigated in our lab. Cytoplasmic segmentation of oligonucleated products
of spontaneous protoplast fusions is of cytological interest since, contrary to findings of other authors (von Aderkas 1992), no preceding karyokinesis was observed. Furthermore, it is apparent that in contrast to free nuclei which are common during early zygotic embryogenesis the cytoskeleton became highly disturbed during the fusion process and has to create individual compartments anew (Stax6n et al. 1994). In rive, the cytoskeleton and nuclei arrangements always remain highly ordered. Comparative investigations on a possible creation of spindles between two nuclei as a prerequisite of cell wall formation and connected aspects might improve our knowledge on the ultrastmctural organization of the cytoplasm.
Acknowledgements. The authors wish to thank Dr. H. Dembny for the initiation and maintenance of the embryogenic suspension cultures. In addition, we would like to acknowledge the contributions of U. Behrendt regarding the development of this protoplast culture system in our lab.
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