Planta
Planta (1988) 176:196 204
9 Springer-Verlag 1988
Acquisition of embryogenic potential in carrot cell-suspension cultures Sacco C. de Vries 1 *, Hilbert Booij 1, Peter Meyerink 1, Gert Huisman 1, H. Dayton Wilde 2, Terry L. Thomas 2' and Ab van Kammen 1 1 Department of Molecular Biology, Agricultural University Wageningen, De Dreijen 11, NL-6703 BC Wageningen, The Netherlands, and 2 Department of Biology, Texas A&M University, College station, Texas 77843, USA
Abstract. Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of PEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [35S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular m R N A from starting cultures with different embryogenic potential and subsequent separation of the [35S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitrotranslation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that * To whom correspondence should be addressed Abbreviations." 2,4-D = 2,4-dichlorophenoxyacetic acid; c D N A = complementary DNA; PAGE = polyacrylamide gel electrophoresis; PEM = proembryogenic mass
the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells. Key words: Cell suspension culture - D a u c u s - Embryogenic potential - Excreted cell factor - Gene expression m R N A (in vitro translation)
Introduction
Since the independent discoveries by Reinert (1958) and Steward et al. (1958) that carrot cells grown in vitro were able to produce somatic embryos, a wealth of data has been acquired on this model system for early plant development (reviewed by Sung et al. 1984 and Nomura and Komamine 1986). Choi and Sung (1984) have shown that the number of differences in protein patterns between somatic embryos, maintained in the absence of the synthetic phytohormone, 2,4-dichlorophenoxyacetic acid (2,4-D) and proliferating cells grown in the presence of 2,4-D is remarkably small. One explanation for this finding could be that many of the genes necessary for somatic-embryo formation are already expressed during unorganized growth in the presence of 2,4-D. A much less studied aspect of somatic embryogenesis is how suspension cultures acquire the potential to generate somatic embryos when placed under suitable conditions, i.e. low cell density in the absence of 2,4-D. So far, the only conclusive evidence on this subject is that a suspension culture must be grown in the presence of 2,4-D in order to become embryogenic. Carrot cell-suspension cultures that possess the potential to produce somatic embryos are invariably characterized by the presence of clusters containing between 10 and 20 small, highly cytoplasmic cells tightly adhering to each other. One or
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
a few of the cells on the surface of these so-called proembryogenic masses (PEMs; Halperin 1966; Halperin and Jensen 1967) will eventually develop into an embryo. The number of PEMs present in different embryogenic carrot suspension cultures varies between 0.1 and 5% of the total number of cells, whereas the remainder of the cells are present in larger aggregates and as single, highly vacuolated cells. The proembryogenic masses are derived under the influence of 2,4-D fi:om single cells of a specific type (Nomura and Komamine 1985) and represent stable intermediates in the morphogenetic pathway leading from single cells to somatic embryos. The single-cell progenitors of PEMs, designated Type I cells, have been isolated manually fiom established suspension cultures (Nomura and Komamine 1985), but in order to study changes at the molecular level during acquisition of embryogenic potential, it is necessary to obtain much larger amounts of suspension cells capable of producing proembryogenic masses. We have developed a culture system in which cells are released from hypocotyl explants directly into 2,4-D-containing liquid medium. In this way, a population of cells can be obtained that will slowly develop proembryogenic masses and thus acquire the potential to produce somatic embryos. We report that in these "starting cultures" the acquisition of embryogenic potential and subsequent formation of somatic embryos is accompanied by only minor changes in gene expression as visualized by two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis of in-vitro translation products and hybridization analysis with a cloned complementary (c)DNA probe. In addition, evidence is presented showing that high-molecularweight, heat-labile, excreted cell factors play a promoting role in the induction of embryogenic potential in carrot suspension cultures. Material and methods Plant material and culture conditions. Daucus carota L. cv. "Flakkese" SG 766 Trophys seeds were generously supplied by Zaadunie B.V., Enkhuizen, The Netherlands. Seeds were surface-sterilized in 70% ethanol for 5 min, 5% (v/v) sodium hypochlorite for 20 rain, washed extensively in sterile, demineralized water and then germinated for 7 d at 25 ~ C in darkness on B5 medium (Gamborg et al. 1968) solidified with 1% agar. Hypocotyl sections without apical meristems were removed from the seedlings and chopped into 2- to 4-ram segments. Hypocotyl tissue (1 2 g fresh weight) was inoculated in 50 ml of 2,4-D containing liquid B5 medium. The starting cultures thus obtained were kept on a rotary shaker at 100 rpm under an 18 h light/6 h dark regime at 25 ~ C. The callused hypocotyl segments were removed with a l-mm-mesh stainless-steel sieve when a sufficiently dense suspension-cell culture had been formed, usually 19 d after culture initiation.
197 Total cell counts were determined with the chromic-aciddispersion method of Sung (1976). For measuring the embryogenic potential of a suspension culture, an aliquot of the cultures was extensively washed with B5 medium without 2,4-D and cultured at an initial density of 5.10 ~ cells.ml-t on 5-cm plates containing B5 medium without 2,4-D. After 12 d, the total number of embryos formed (all stages of development) was determined. The embryogenic potential is expressed as the number of embryos formed by 10 ~ suspension cells. High-density embryogenic suspension cultures were maintained in B5 medium with 2 gM 2,4-D at a 14-d subculture cycle by inoculating 2 ml of packed cell volume in 50 ml of medium, corresponding to an initial cell density of approx. 106 cells.ml-1. Proembryogenic masses (48-120 ~m) were obtained from a 7-d embryogenic suspension culture by filtration through 120-I.tm and 48-gin nylon meshes. Unless stated otherwise, all cultures contained over 95 % viable cells as determined by fluorescein diacetate/Lisamin Green staining.
Preparation of cell-free conditioned medium. Cell-free medium conditioned by established embryogenic suspension cultures was obtained from high-density suspension cultures, 7 d after the last subculture, by centrifugation at 300-g, filtration of the supernatant through Whatman (Springfield Hill, UK) 1 M M paper and filter sterilization through a 0.22 gm Milipore filter. High-molecular-weight fractions of conditioned media were obtained by pressure dialysis through an Amicon (Oosterhout, The Netherlands) YM5000 membrane yielding 100- to 500times-concentrated preparations (concentrate) and a filtrate devoid of proteins and polysaccharides. Gene-expression analysis. Total R N A was extracted from frozen plant tissue according to De Vries et al. (1982) except that the extraction buffer contained 0.1 M LiC1; 10 mM ethylenediaminetetraacetic acid (EDTA); 1% (w/v sodium dodecyl sulfate (SDS) 0.1 M 2-amino-2-(hydroxymethyl)- 1,3-propanediol (Tris)-HC1 (pH 9.0). In-vitro translation in the presence of [35S]methionine and subsequent two-dimensional PAGE analysis of the labeled products were as described (De Vries et al. 1982). For R N A gel blots, total R N A was denatured with glyoxal and dimethyl sulfoxide (DMSO), fractionated on a 1% agarose gel, and transferred to nitrocellulose (Thomas 1980). The c D N A insert of De3 was gel purified and labeled by nick translation (Maniatis et al. 1982); hybridization was as described (Allen et al. 1985).
Results
Characterization of the starting-culture system. Since the aim of the culture system was to obtain a cell population in which the transition from nonembryogenic cells into embryogenic cells could be monitored, several parameters that might influence the development of the embryogenic potential have been considered (Fig. 1). Cells referred to as being embryogenic are those that will produce somatic embryos following dilution and withdrawal of 2,4D. An important aspect of the starting-culture system is that suspension cells newly liberated from the explant tissue are essentially non-embryogenic up to day 15. The cells still adhering to the explant (Fig. 2A) are, similar to the free-floating suspension cells, completely non-embryogenic, although
198
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
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Fig. 1. Development of embryogenic potential in newly started carrot cell-suspension cultures. Open symbols represent the number of suspension cells.ml -~ in cultures containing 0.45 gM (D-m), 2 gM (o-o) and 4.5 pM (A-A) of 2,4-D. Closed symbols represent the embryogenic potential expressed in the number of somatic embryos (all stages) produced per 104 suspension cells as measured in newly started cultures containing 0.45 pM ( l - n ) , 2 llM (o-o) and 4.5 gM ( i - i ) of 2,4-D. The culture inoculated in 0.45 pM 2,4-D was not followed after day 30 (stippled line). The arrow at day 19 indicates the removal of the hypocotyl explant; while the bar spanning days 14~21 denotes the period in which the explant can be removed without affecting the growth of the culture. The second arrow represents the time of the first subculture, performed by dilution to ]0 6 cells.ml- ~ with fresh B5 medium with 2,4-D. Subsequent subcultures were at 14-d intervals as indicated by the vertical stippled lines. The stippled bar represents the embryogenic potential 7 d after the last subculture of an established embryogenic culture of the same cultivar
root formation has been observed from these cells upon transfer of hypocotyl explants to B5 medium without hormones (data not shown). At day 19, when the hypocotyl explants were removed, the starting suspension has reached a density of about 1 0 6 cells.ml -~. The cells at this stage are derived mainly from the callus tissue formed at the cut surfaces of the hypocotyl explants and are mostly vacuolated single cells and small clusters showing numerous cell divisions (Fig. 2 B). The capacity of the liberated, and now proliferating, suspension cells to produce embryos became apparent from day 20 onwards, although the number of embryos produced was only about 3-10% of the value obtained for an established embryogenic culture. At
Fig. 2A-C. Morphology of cells present in carrot suspension cultures directly initiated from hypocotyl explants. A Hypocotyl explant with callus cells growing on wounded surface. B Suspension cells 19 d after culture initiation. The difference between the large vacuolated cells and a proembryogenic mass (arrowhead) is clearly visible. C Suspension cells 40 d after culture initiation. More proembryogenic masses are now present in the culture (arrowheads) concurrent with the increase in embryogenic potential in these cultures. Bars = 48 IxM
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
the same time, a new, multicellular structure appeared in the culture, forming clusters of between 10 and 20 small highly cytoplasmic cells tightly adhering to each other (Fig. 2 B, C). In both their physical characteristics and embryogenic capacity, these clusters resemble the PElVis described by Halperin (1966). The number of PEMs present in the starting cultures reflected quite closely the measured embryogenic potential. Furthermore, since it was observed that somatic embryos that develop in an established embryogenic suspension culture originate predominantly from these characteristic clusters of cells, we conclude that the appearance of the PEMs marks the beginning of the embryogenie potential in newly started carrot cultures. Further inspection of Fig. I shows that the bulk of the embryogenic capacity is obtained after day 50 and reaches a maximum after more than 2.5 months in culture. In the period after day 50, the average growth rate of the starting cultures becomes similar to those normally observed in established cultures. To verify whether the concentration of 2,4-D is rate limiting in the acquisition of embryogenic potential, cultures were started from hypocotyls at 0.45, 2 and 4.5 gM of 2,4-D. The results in Fig. 1 show that at the lowest 2,4-D concentration examined the culture is not stable and starts dying off after day 30. Cultures initiated in twice the optimal 2,4-D concentration do not acquire embryogenic potential faster or to a higher level. Thus, it seems above a certain threshold concentration the development of embryogenic potential is independent of the 2,4-D concentration. The horizontal bar in Fig. I represents the period in which the hypocotyl explant must be removed. Withdrawing the explant before day 14 resulted in poorly growing suspensions with low embryogenic potential; whereas removal after day 21 yielded equally poor growing suspensions with many dead cells present. Apparently, between days 14 and 21 a sufficiently high portion of the suspension must have started to divide before the concentration of certain critical medium components becomes too low to sustain further growth, or the concentration of growth-inhibiting factors produced by the explant tissue as a result of wounding becomes to high.
Excreted cell factors promote the acquisition of embryogenic potential. Since the data presented in Fig. I indicate that acquisition of embryogenic potential requires a more-or-less fixed period of active growth at high cell density, we asked whether medium, preconditioned by an established embryogenic suspension, contains factors that are re-
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Fig. 3. Effect of conditioned medium on development of embryogenic potential in newly started carrot cell-suspension cultures. Open symbols represent the number of suspension cells. ml 1 in a culture containing 2 ~tM of 2,4-D. Closed symbols represent the embryogenic potential expressed as the number of embryos (all stages) produced by 104 suspension cells as measured in newly started cultures with the following additions: no addition (o o); addition at day 19 of 50% (v/v) cell-free medium conditioned by a 7-d established embryogenic suspension culture ( I - I ) ; addition at day 19 of a 50% (v/v) equivalent high-molecular-weight fraction (over 5 kDa) obtained from the cell-free conditioned medium of a 7-d established embryogenic suspension culture (A-A); addition at day 19 of a 50% (v/v) equivalent high-molecular-weight fraction, treated for 10 min at 100~ C, obtained from cell-free medium conditioned by a 7-d-old established embryogenic suspension culture ( , T); addition at day 19 of 50% (v/v) cell-free conditioned medium, devoid of material over 5 kDa, obtained from a 7-d established embryogenic suspension culture (• x). The arrow at day 19 indicates the removal of the hypocotyl explant in all cultures, while at day 30 (second arrow) all cultures were transferred to fresh B5 medium with 2 gM 2,4-D
quired in order to produce large numbers of PEMs. Half of the medium in a standard starting culture was replaced by filter-sterilized, conditioned medium from a 7-d embryogenic suspension culture concomitant with removal of the hypocotyl explant at day 19. Further subculturing starting at day 30 was performed with fresh medium only. This treatment resulted in a dramatic increase in the number of proembryogenic masses observed and hence in embryogenic potential (Fig. 3). The levels attained were similar to levels normally observed at least I month later in untreated control cultures. Addition of 100- to 500-fold Amicon-concentrated conditioned medium, containing only material larger than 5 kDa produced the same promoting effect as unfractionated conditioned medium; heat treatment of this material destroyed the effect. Since conditioned medium devoid of the high-molecular-weight material did not give a promoting effect (Fig. 3), the possibility that phytohormones or other low-molecular-weight inducers excreted by the cells are the causative agents can
200
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
During the acquisition of embryogenic potential there is a gradual increase in the amount of a 72kDa extracellular protein that is also absent in media conditioned by somatic embryos. Taken together, the pattern of [35S]methionine-labeled extracellular polypeptides produced during the acquisition of embryogenic potential becomes increasingly complex in parallel to the increase in embryogenic potential.
Gene expression during development of embryogenic potential. The pattern of gene expression observed
Fig. 4. Extracellular polypeptides labeled with [35S]methionine at different times in a starting culture of carrot suspension cells and separated by 12.5% PAGE, For comparison, the extracellular polypeptides produced by an established high-density suspension culture and the embryos and its callus derived from such a culture are also shown
be excluded. None of the described culture treatments gave significant alterations of the macroscopic growth rates of the cultures when compared to the unsupplemented culture (data not shown).
Synthesis of extracellularpolypeptides during acquisition of embryogenic potential. Figure 4 shows the [35S] methionine-labeled proteins that are excreted into the medium of starting cultures at different times after inoculation. The extracellular-protein patterns of an established, high-density embryo~ genic culture and of somatic embryos produced from this culture are also shown. Before embryogenic potential is acquired (see Fig. 1 and 4, day 4), the pattern is surprisingly like that of an embryo culture, e.g. fairly high amounts of a 90-kDa protein. There appears to be a sudden increase at day 9 of a 27 kDa polypeptide which is present in embryogenic suspension cultures and is absent or greatly reduced during somatic embryogenesis.
during transition of the explant cells into an embryogenic suspension culture and in the somatic embryos generated from these cultures was analyzed by in-vitro translation of total cellular m R N A followed by two-dimensional PAGE analysis. Comparison of the patterns obtained from hypocotyl explants at day 14 (Fig. 5A), non-embryogenic suspension cells from a starting culture at day 14 (Fig. 5B), embryogenic suspension cells (Fig. 5C) and somatic embryos (Fig. 5D) show that the abundant m R N A populations visualized this way are quite similar. There are, however, a few notable differences observed in the translation products of the different developmental states examined. By day 14, callus formation at the cut surfaces of the hypocotyl explants is well underway. Comparison of in-vitrotranslation products from these explants and from suspension cells released from the explants at day 14 shows four spots are represented at lower levels in the released cell sample. Two products are present in the released cells that are not found in hypocotyl explant tissue (arrowheads in Fig. 5 A, B). At day 61, when embryogenic potential has developed in the starting culture, four other products (arrowheads in Fig. 5 C, open circles in Fig. 5A, B) have appeared that are not present in hypocotyl explants or in 14-d starting cultures. These in-vitro-translation products could represent genes involved in the acquisition of embryogenic potential. One product has disappeared from the pattern at day 61 (arrowheads in Fig. 5A, B and open circle in 5 C, D). Upon initiation of embryogenesis by dilution of proembryogenic masses in 2,4-D free medium, two products appear (arrowheads in Fig. 5 D, open circles in 5A, B, C) that are present only in somatic embryos; while six products disappear (arrowheads in Fig. 5A, B, C open circles in 5D). Three of the four in-vitrotranslation products that appeared first in 61-d embryogenic suspension cells increased considerably in somatic embryos (arrowheads in Fig. 5C, D, open circles in 5 A, B). One product is present only
S.C. de Vries et al. : Acquisitionof embryogenicpotential in carrot
201
Fig. 5A-D. Fluorographs of [35S]methionine-labeledin-vitro-translationproducts obtained from a carrot-mRNA-directed wheatgerm extract and separated by two-dimensionalPAGE as outlined in methods. A Hypocotylexplants after 14 d immersion in B5 mediumwith 2 ~tM2,4-D. B Non-embryogenicsuspension cells liberated from the explants at day 14. C Embryogenicsuspension cells at day 61 after initiation. D Somatic embryos. Arrowheads indicate in-vitro-translationproducts present in one pattern and absent in others (open circles)
in embryogenic cultures (arrowhead in Fig. 5C, open circles in 5A, B, C).
Expression of an embryo-regulated gene is associated with the acquisition of embrvogenic potential. Differential screening of a 2gtll embryo cDNA library with somatic embryo and suspension polyadenylated R N A has yielded several cDNA recombinants that on subsequent analysis showed a higher level of expression in somatic embryos than in proliferating cells of the embryogenic carrot line W D I . One of these cDNA sequences, designated Dc3, was highly expressed as m R N A in PEMs and somatic embryos (Thomas and Wilde 1985, 1987). The utility of Dc3 as a marker for the acquisition of embryogenic potential was further explored by
using it to probe R N A gel blots containing total RNA obtained from the Flakkese SG766 starting cell suspensions. The results of one experiment are shown in Fig. 6 and demonstrate that a 750-nucleotide transcript complementary to Dc3 is present in somatic embryos and to a much lower extent in unfractionated 51 d embryogenic starting cultures. Dc3 m R N A is undetectable in the non-embryogenic cells of 14-d and 22-d starting cultures. From this experiment, it is apparent that Dc3 is expressed only when PEMs are present in the starting cultures. Furthermore, these results clearly demonstrate that Dc3 expression is not a cellular response to culturing in the presence of 2,4-D. The pattern of expression demonstrated by Dc3 strongly indicates that proembryogenic masses, being an
202
Fig. 6. Patterns of RNA expression of an embryo-regulated cDNA, Dc3. Hybridization of Dc3 to a gel blot containing total RNA (8 gg per lane) extracted from carrot suspension cells at different times during acquisition of embryogenic potential. nt, nucleotides
essential prerequisite for somatic embryogenesis, can be considered as the first distinct and differentiated stage of somatic embryogenesis. Discussion
Somatic embryogenesis in Daucus carota has been studied extensively from a physiological point of view (Nomura and Komamine 1986), and although the molecular mechanisms that determine and control somatic embryogenesis are not yet understood, progress has been made in understanding the factors involved in the initiation and maintenance of this developmental pathway. Nomura and Komamine (1985) demonstrated that morphologically distinct single cells exist in suspension cultures maintained at high density and in the presence of high 2,4-D concentrations. These cells, designated Type 1 cells, are capable of developing into proembryogenic masses after exposure to low levels of 2,4-D. It is unclear at present whether these embryogenic cells derive from a common precursor cell type already present in the original explant or, alternatively, are continuously formed de novo from non-embryogenic cells. Subsequent events of the somatic embryogenesis pathway occur in the absence of exogenous 2,4-D and result in the gross morphogenetic changes leading to the formation of somatic embryos. In carrot, as well as in many other plant species, the synthetic auxin, 2,4-D, is the most efficient inducer of embryogenic potential (Ammirato 1983; Sung et al. 1984). This auxin analogue facilitates the development of somatic embryos up to the
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
globular stage, but it acts as a potent inhibitor of further development (Borkird et al. 1986). Virtually nothing is known about the molecular mechanisms by which 2,4-D induces embryogenic potential in plant tissue cultures. The experiments presented here demonstrate that carrot cells newly liberated from explant tissue have not yet acquired the capacity to produce somatic embryos. Although no attempt has been made to isolate the single cell precursors, or Type 1 cells, for proembryogenic masses during this period, it seems logical to assume that they are formed during the first two weeks of a starting culture. This period can be thought of as the time needed for cells to dedifferentiate and to obtain the competence to give rise to embryogenic cells, perhaps similar to the period in which Convolvulus leaf explants acquire the competence to produce shoots (Christianson and Warnick 1984, 1985). A stimulatory effect of conditioned medium on somatic embryogenesis has been demonstrated in carrot by Hari (1980) and Smith and Sung (1985). However, few attempts have been made to identify the responsible factors; although during regeneration of barley microspores, modified phytohormones have been found to be the active component (K6hler and Wenzel 1985). In contrast, our results indicate that the components of conditioned medium, active in the acquisition of embryogenic potential in starting cultures, are high-molecular-weight factors. This excludes the possibility that phytohormones, oligosaccharides (Tran Thanh Van et al. 1985), polyamines (Fienberg et al. 1984) or other low-molecular-weight components of conditioned medium are the causative agents. Because of the heat-lability of the effect, we tentatively conclude that excreted polypeptide(s) are the active components. Recently, Satoh et al. (1986) described a carrot extracellular glycoprotein of 65 kDa that appeared in the medium of somatic embryos only. In our analysis, however, we have not encountered a protein of identical molecular weight and behavior. A wide variety of lytic enzymes such as peroxidases (Chibbar et al. 1984), proteases and phosphatases (Wink 1984) are present in conditioned medium, but presently it is unknown whether any of these known excreted enzymes is involved in determination of embryogenic potential in carrot. It is noteworthy that a comparison of 3SS-labeled extracellular polypeptides produced during the acquisition of embryogenic potential shows a considerable increase in complexity parallel to the increasing embryogenic potential. The analysis of translational profiles of m R N A isolated from hypocotyl explants, starting suspen-
s.c. de Vries et al. : Acquisition of embryogenic potential in carrot sion cells, e m b r y o s a n d proliferating callus cells c o n f i r m s previous o b s e r v a t i o n s (Sung a n d O k i m o to 1981, 1983) t h a t differences in gene expression between o r g a n i z e d cells, like s o m a t i c e m b r y o s , a n d u n o r g a n i z e d cells, like callus a n d suspension cultures, are r e m a r k a b l y small a n d involve less t h a n ten i n - v i t r o - t r a n s l a t i o n p r o d u c t s out o f the a p p r o x imately 300 spots resolved on t w o - d i m e n s i o n a l gels between p H 4.5 a n d 7.5. The s a m e result was obtained if the t r a n s l a t i o n a l profiles o f the original explants a n d the derived suspension cells are c o m pared, indicating t h a t gross changes in the expression o f genes e n c o d i n g a b u n d a n t p o l y p e p t i d e s d o n o t o c c u r during dedifferentiation, induction o f embryogenic potential and formation of somatic e m b r y o s . M o s t differences were detected between s o m a t i c e m b r y o s a n d suspension cells f r o m starting cultures w i t h o u t p r o e m b r y o g e n i c masses. T h e gene c o r r e s p o n d i n g to the c D N A r e c o m binant, Dc3, is expressed in cultures t h a t c o n t a i n only p r o e m b r y o g e n i c masses or s o m a t i c e m b r y o s . This result, along with o b s e r v a t i o n s on the p a t terns o f gone expression in P E M s a n d s o m a t i c embryos, provides a m o l e c u l a r basis to the c o n c e p t t h a t specific genes involved in s o m a t i c e m b r y o g e n esis are already expressed long before m o r p h o l o g i cally discernible s o m a t i c e m b r y o s are present. T h e implications o f this finding are t h a t d e t e r m i n a t i o n o f cells destined to p r o d u c e s o m a t i c e m b r y o s takes place m u c h earlier t h a n previously a s s u m e d a n d u n d e r conditions t h a t inhibit the gross m o r p h o l o g ical changes associated with s o m a t i c e m b r y o g e n e sis. T h e availability o f the starting-culture system described here a n d m o l e c u l a r m a r k e r s such as Dc3 n o w allow the analysis o f the onset o f these e m b r y onic gene-expression p r o g r a m s . F u r t h e r m o r e , the starting cultures will be useful in the identification o f genes directly involved in the acquisition o f emb r y o g e n i c potential in c a r r o t suspension cultures. S o m e o f these genes are p r o b a b l y expressed u n d e r the influence o f 2,4-D a n d m a y encode extracellular proteins. We thank Piet Madern and Reindert de Fluiter for artwork and photography and Hedy Adriaansz, Rilla Eaton and Sandra Garcia for typing the manuscript. This work was supported in part by USDA Competitive Grants 84CRCR-I-1391 and 86CRCR-1-2143 awarded to T.L.T. Dayl:on Wilde is the recipient of a W.R. Grace fellowship.
References
Allen, R.D., Nessler, C.L., Thomas, T.L. (1985) Developmental expression of sunflower 11S storage protein genes. Plant Mol. Biol. 5, 165 173 Ammirato, P.V. (1983) Embryogenesis. In: Handbook of plant
203 cell culture, vol. I, pp. 88 123, Evans, D.A., Sharp, W.R., Ammirato, P.V., Yamada, Y. eds. Macmillan, New York Borkird, C., Choi, J.H., Sung, Z.R. (1986) Effect of 2,4-dichlorophenoxyacetic acid on the expression of embryogenic program in carrot. Plant Physiol 81, 1143-1146 Chibbar, R.N., Cella, R., Albani, D., van Huystee, R.B. (1984) The growth and peroxidase synthesis by two carrot cell lines. J. Exp. Bot. 35, 1846-1852 Choi, J.H. Sung, Z.R. (1984) Two-dimensional gel analysis of carrot somatic embryonic proteins. Plant Mol. Biol. Reporter 2, 19-25 Christianson, M.L., Warnick, D.A. (1984) Phenocritical times in the process of in vitro shoot organogenesis. Dev. Biol. 101, 382-390 Christianson, J.L., Warnick, D.A. (1985) Temporal requirements for phytohormone balance in the control of organogenesis in vitro. Dev. Biol. 112, 494-497 De Vries, S.C., Springer, J., Wessels, J.G.H. (1982) Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings. Planta 156, 129-135
Fienberg, A.A., Choi, J.H., Lubich, W.P., Sung, Z.R. (1984) Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture. Planta 162, 532-539 Gamborg, O.L., Miller, R.A., Ojima, K. (1968) Plant cell cultures: I. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell. Res. 50, 151-158 Halperin, W. (1966) Alternative morphogenetic events in cell suspensions. Am. J. Bot. 53, 443-453 Halperin, W., Jensen, W.A. (1967) Ultrastructural changes during growth and embryogenesis in carrot cell cultures. J. U1trastruc. Res_ 18, 428-443 Hari, V. (1980) Effect of cell density changes and conditioned media on carrot cell embryogenesis. Z. Pflanzenphysiol. 96, 227-231 K6hler, F., Wenzel, G. (1985) Regeneration of isolated barley microspores in conditioned media and trials to characterize the responsible factor. J. Plant Physiol. 121, 181 191 Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Upton, N.Y., USA Nomura, K., Komanine, A. (1985) Identification and isolation of single cells that produce somatic embryos at a high frequency in carrot suspension cultures. Plant Physiol. 79, 988 991 Nomura, K., Komamine, A. (1986) Molecular mechanisms of somatic embryogenesis. Oxford Surveys Plant Mol. Cell. Biol. 3, 456-466 Reinert, J. (1958) Morphogenese und ihre Kontrolle an Gewebekulturen aus Carotten. Naturwissenschaften 45, 344-345 Satoh, S., Kamada, H., Harada, H., Fnji, T. (1986) Auxincontrolled glycoprotein release into the medium of embryogenic carrot cells. Plant Physiol. 81,931-933 Smith, J.A., Sung, Z.R. (1985) Increase of regeneration of plant cells by cross feeding with regenerating Daneus carota cells. In: Somatic embryogenesis, pp. 133-136, Terzi, M., Pitto, L., Sung, Z.R., eds. IPRA, Rome Steward, F.C., Mapes, M.O., Hears, K. (1958) Growth and organized development of cultured ceils: II. Organization in cultures grown from freely suspended ceils. Am. J. Bot. 45, 705-708 Sung, Z.R. (1976) Turbidimetric determination of plant cell culture growth. Plant Physiol. 57, 460-462 Sung, Z.R., Feinberg, A., Cborneau, R., Borkird, C., Furner, I., Smith, J., Terzi, M., LoSchiavo, F., Giuliano, G., Pitto, L., Nuti-Ronchi, V. (1984) Developmental biology of em-
204 bryogenesis from carrot culture. Plant Mol. Biol. Rep. 2, 3-14 Sung, Z.R., Okimoto, R. (1981) Embryonic proteins in somatic embryos of carrot. Proc. Natl. Acad. Sci. USA. 78, 3683 3687 Sung, Z.R., Okimoto, R. (1983) Coordinate gene expression during somatic embryogenesis in carrots. Proc. Natl. Acad. Sci. USA 80, 2661-2665 Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201 5206 Thomas, T.L., Wilde, H.D. (1985) Analysis of gene expression in carrot somatic embryos. In: Somatic embryogenesis, pp. 7285, Terzi, J., Pitto, L., Sung, Z.R., eds. IPRA, Rome
Note added in proof
Recently we have also demonstrated that extracellular proteins are involved in the development of somatic embryos from proembryogenic masses. (S.C. de Vries et al. Genes & Development 2, 462-476, 1988)
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot Thomas, T.L., Wilde, D. (1987) Analysis of carrot somatic embryo gene expression programs (Proc. VI Int. Congr. Plant Celt and Tissue Culture) pp. 83 93. Alan R. Liss, New York Tran Thanh Van, K., Tonbart, P., Cousson, A., Darvill, A.G., Gollin, D.J., Chelf, P. Albersheim, P. (1985) Manipulation of the morphogenetic pathways of tobacco by oligosaccarins. Nature 314, 615-617 Wink, M. (1984) Evidence for an extracellular lytic compartment of plant cell suspension cultures: the cell culture medium. Naturwissenschaften 71, 635-638
Received 29 October/987; accepted 31 March 1988
Planta (1988) 176:196 204
9 Springer-Verlag 1988
Acquisition of embryogenic potential in carrot cell-suspension cultures Sacco C. de Vries 1 *, Hilbert Booij 1, Peter Meyerink 1, Gert Huisman 1, H. Dayton Wilde 2, Terry L. Thomas 2' and Ab van Kammen 1 1 Department of Molecular Biology, Agricultural University Wageningen, De Dreijen 11, NL-6703 BC Wageningen, The Netherlands, and 2 Department of Biology, Texas A&M University, College station, Texas 77843, USA
Abstract. Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of PEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [35S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular m R N A from starting cultures with different embryogenic potential and subsequent separation of the [35S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitrotranslation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that * To whom correspondence should be addressed Abbreviations." 2,4-D = 2,4-dichlorophenoxyacetic acid; c D N A = complementary DNA; PAGE = polyacrylamide gel electrophoresis; PEM = proembryogenic mass
the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells. Key words: Cell suspension culture - D a u c u s - Embryogenic potential - Excreted cell factor - Gene expression m R N A (in vitro translation)
Introduction
Since the independent discoveries by Reinert (1958) and Steward et al. (1958) that carrot cells grown in vitro were able to produce somatic embryos, a wealth of data has been acquired on this model system for early plant development (reviewed by Sung et al. 1984 and Nomura and Komamine 1986). Choi and Sung (1984) have shown that the number of differences in protein patterns between somatic embryos, maintained in the absence of the synthetic phytohormone, 2,4-dichlorophenoxyacetic acid (2,4-D) and proliferating cells grown in the presence of 2,4-D is remarkably small. One explanation for this finding could be that many of the genes necessary for somatic-embryo formation are already expressed during unorganized growth in the presence of 2,4-D. A much less studied aspect of somatic embryogenesis is how suspension cultures acquire the potential to generate somatic embryos when placed under suitable conditions, i.e. low cell density in the absence of 2,4-D. So far, the only conclusive evidence on this subject is that a suspension culture must be grown in the presence of 2,4-D in order to become embryogenic. Carrot cell-suspension cultures that possess the potential to produce somatic embryos are invariably characterized by the presence of clusters containing between 10 and 20 small, highly cytoplasmic cells tightly adhering to each other. One or
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
a few of the cells on the surface of these so-called proembryogenic masses (PEMs; Halperin 1966; Halperin and Jensen 1967) will eventually develop into an embryo. The number of PEMs present in different embryogenic carrot suspension cultures varies between 0.1 and 5% of the total number of cells, whereas the remainder of the cells are present in larger aggregates and as single, highly vacuolated cells. The proembryogenic masses are derived under the influence of 2,4-D fi:om single cells of a specific type (Nomura and Komamine 1985) and represent stable intermediates in the morphogenetic pathway leading from single cells to somatic embryos. The single-cell progenitors of PEMs, designated Type I cells, have been isolated manually fiom established suspension cultures (Nomura and Komamine 1985), but in order to study changes at the molecular level during acquisition of embryogenic potential, it is necessary to obtain much larger amounts of suspension cells capable of producing proembryogenic masses. We have developed a culture system in which cells are released from hypocotyl explants directly into 2,4-D-containing liquid medium. In this way, a population of cells can be obtained that will slowly develop proembryogenic masses and thus acquire the potential to produce somatic embryos. We report that in these "starting cultures" the acquisition of embryogenic potential and subsequent formation of somatic embryos is accompanied by only minor changes in gene expression as visualized by two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis of in-vitro translation products and hybridization analysis with a cloned complementary (c)DNA probe. In addition, evidence is presented showing that high-molecularweight, heat-labile, excreted cell factors play a promoting role in the induction of embryogenic potential in carrot suspension cultures. Material and methods Plant material and culture conditions. Daucus carota L. cv. "Flakkese" SG 766 Trophys seeds were generously supplied by Zaadunie B.V., Enkhuizen, The Netherlands. Seeds were surface-sterilized in 70% ethanol for 5 min, 5% (v/v) sodium hypochlorite for 20 rain, washed extensively in sterile, demineralized water and then germinated for 7 d at 25 ~ C in darkness on B5 medium (Gamborg et al. 1968) solidified with 1% agar. Hypocotyl sections without apical meristems were removed from the seedlings and chopped into 2- to 4-ram segments. Hypocotyl tissue (1 2 g fresh weight) was inoculated in 50 ml of 2,4-D containing liquid B5 medium. The starting cultures thus obtained were kept on a rotary shaker at 100 rpm under an 18 h light/6 h dark regime at 25 ~ C. The callused hypocotyl segments were removed with a l-mm-mesh stainless-steel sieve when a sufficiently dense suspension-cell culture had been formed, usually 19 d after culture initiation.
197 Total cell counts were determined with the chromic-aciddispersion method of Sung (1976). For measuring the embryogenic potential of a suspension culture, an aliquot of the cultures was extensively washed with B5 medium without 2,4-D and cultured at an initial density of 5.10 ~ cells.ml-t on 5-cm plates containing B5 medium without 2,4-D. After 12 d, the total number of embryos formed (all stages of development) was determined. The embryogenic potential is expressed as the number of embryos formed by 10 ~ suspension cells. High-density embryogenic suspension cultures were maintained in B5 medium with 2 gM 2,4-D at a 14-d subculture cycle by inoculating 2 ml of packed cell volume in 50 ml of medium, corresponding to an initial cell density of approx. 106 cells.ml-1. Proembryogenic masses (48-120 ~m) were obtained from a 7-d embryogenic suspension culture by filtration through 120-I.tm and 48-gin nylon meshes. Unless stated otherwise, all cultures contained over 95 % viable cells as determined by fluorescein diacetate/Lisamin Green staining.
Preparation of cell-free conditioned medium. Cell-free medium conditioned by established embryogenic suspension cultures was obtained from high-density suspension cultures, 7 d after the last subculture, by centrifugation at 300-g, filtration of the supernatant through Whatman (Springfield Hill, UK) 1 M M paper and filter sterilization through a 0.22 gm Milipore filter. High-molecular-weight fractions of conditioned media were obtained by pressure dialysis through an Amicon (Oosterhout, The Netherlands) YM5000 membrane yielding 100- to 500times-concentrated preparations (concentrate) and a filtrate devoid of proteins and polysaccharides. Gene-expression analysis. Total R N A was extracted from frozen plant tissue according to De Vries et al. (1982) except that the extraction buffer contained 0.1 M LiC1; 10 mM ethylenediaminetetraacetic acid (EDTA); 1% (w/v sodium dodecyl sulfate (SDS) 0.1 M 2-amino-2-(hydroxymethyl)- 1,3-propanediol (Tris)-HC1 (pH 9.0). In-vitro translation in the presence of [35S]methionine and subsequent two-dimensional PAGE analysis of the labeled products were as described (De Vries et al. 1982). For R N A gel blots, total R N A was denatured with glyoxal and dimethyl sulfoxide (DMSO), fractionated on a 1% agarose gel, and transferred to nitrocellulose (Thomas 1980). The c D N A insert of De3 was gel purified and labeled by nick translation (Maniatis et al. 1982); hybridization was as described (Allen et al. 1985).
Results
Characterization of the starting-culture system. Since the aim of the culture system was to obtain a cell population in which the transition from nonembryogenic cells into embryogenic cells could be monitored, several parameters that might influence the development of the embryogenic potential have been considered (Fig. 1). Cells referred to as being embryogenic are those that will produce somatic embryos following dilution and withdrawal of 2,4D. An important aspect of the starting-culture system is that suspension cells newly liberated from the explant tissue are essentially non-embryogenic up to day 15. The cells still adhering to the explant (Fig. 2A) are, similar to the free-floating suspension cells, completely non-embryogenic, although
198
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
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.
. 50
.
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Fig. 1. Development of embryogenic potential in newly started carrot cell-suspension cultures. Open symbols represent the number of suspension cells.ml -~ in cultures containing 0.45 gM (D-m), 2 gM (o-o) and 4.5 pM (A-A) of 2,4-D. Closed symbols represent the embryogenic potential expressed in the number of somatic embryos (all stages) produced per 104 suspension cells as measured in newly started cultures containing 0.45 pM ( l - n ) , 2 llM (o-o) and 4.5 gM ( i - i ) of 2,4-D. The culture inoculated in 0.45 pM 2,4-D was not followed after day 30 (stippled line). The arrow at day 19 indicates the removal of the hypocotyl explant; while the bar spanning days 14~21 denotes the period in which the explant can be removed without affecting the growth of the culture. The second arrow represents the time of the first subculture, performed by dilution to ]0 6 cells.ml- ~ with fresh B5 medium with 2,4-D. Subsequent subcultures were at 14-d intervals as indicated by the vertical stippled lines. The stippled bar represents the embryogenic potential 7 d after the last subculture of an established embryogenic culture of the same cultivar
root formation has been observed from these cells upon transfer of hypocotyl explants to B5 medium without hormones (data not shown). At day 19, when the hypocotyl explants were removed, the starting suspension has reached a density of about 1 0 6 cells.ml -~. The cells at this stage are derived mainly from the callus tissue formed at the cut surfaces of the hypocotyl explants and are mostly vacuolated single cells and small clusters showing numerous cell divisions (Fig. 2 B). The capacity of the liberated, and now proliferating, suspension cells to produce embryos became apparent from day 20 onwards, although the number of embryos produced was only about 3-10% of the value obtained for an established embryogenic culture. At
Fig. 2A-C. Morphology of cells present in carrot suspension cultures directly initiated from hypocotyl explants. A Hypocotyl explant with callus cells growing on wounded surface. B Suspension cells 19 d after culture initiation. The difference between the large vacuolated cells and a proembryogenic mass (arrowhead) is clearly visible. C Suspension cells 40 d after culture initiation. More proembryogenic masses are now present in the culture (arrowheads) concurrent with the increase in embryogenic potential in these cultures. Bars = 48 IxM
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
the same time, a new, multicellular structure appeared in the culture, forming clusters of between 10 and 20 small highly cytoplasmic cells tightly adhering to each other (Fig. 2 B, C). In both their physical characteristics and embryogenic capacity, these clusters resemble the PElVis described by Halperin (1966). The number of PEMs present in the starting cultures reflected quite closely the measured embryogenic potential. Furthermore, since it was observed that somatic embryos that develop in an established embryogenic suspension culture originate predominantly from these characteristic clusters of cells, we conclude that the appearance of the PEMs marks the beginning of the embryogenie potential in newly started carrot cultures. Further inspection of Fig. I shows that the bulk of the embryogenic capacity is obtained after day 50 and reaches a maximum after more than 2.5 months in culture. In the period after day 50, the average growth rate of the starting cultures becomes similar to those normally observed in established cultures. To verify whether the concentration of 2,4-D is rate limiting in the acquisition of embryogenic potential, cultures were started from hypocotyls at 0.45, 2 and 4.5 gM of 2,4-D. The results in Fig. 1 show that at the lowest 2,4-D concentration examined the culture is not stable and starts dying off after day 30. Cultures initiated in twice the optimal 2,4-D concentration do not acquire embryogenic potential faster or to a higher level. Thus, it seems above a certain threshold concentration the development of embryogenic potential is independent of the 2,4-D concentration. The horizontal bar in Fig. I represents the period in which the hypocotyl explant must be removed. Withdrawing the explant before day 14 resulted in poorly growing suspensions with low embryogenic potential; whereas removal after day 21 yielded equally poor growing suspensions with many dead cells present. Apparently, between days 14 and 21 a sufficiently high portion of the suspension must have started to divide before the concentration of certain critical medium components becomes too low to sustain further growth, or the concentration of growth-inhibiting factors produced by the explant tissue as a result of wounding becomes to high.
Excreted cell factors promote the acquisition of embryogenic potential. Since the data presented in Fig. I indicate that acquisition of embryogenic potential requires a more-or-less fixed period of active growth at high cell density, we asked whether medium, preconditioned by an established embryogenic suspension, contains factors that are re-
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30
40 days
50
60
70
80
Fig. 3. Effect of conditioned medium on development of embryogenic potential in newly started carrot cell-suspension cultures. Open symbols represent the number of suspension cells. ml 1 in a culture containing 2 ~tM of 2,4-D. Closed symbols represent the embryogenic potential expressed as the number of embryos (all stages) produced by 104 suspension cells as measured in newly started cultures with the following additions: no addition (o o); addition at day 19 of 50% (v/v) cell-free medium conditioned by a 7-d established embryogenic suspension culture ( I - I ) ; addition at day 19 of a 50% (v/v) equivalent high-molecular-weight fraction (over 5 kDa) obtained from the cell-free conditioned medium of a 7-d established embryogenic suspension culture (A-A); addition at day 19 of a 50% (v/v) equivalent high-molecular-weight fraction, treated for 10 min at 100~ C, obtained from cell-free medium conditioned by a 7-d-old established embryogenic suspension culture ( , T); addition at day 19 of 50% (v/v) cell-free conditioned medium, devoid of material over 5 kDa, obtained from a 7-d established embryogenic suspension culture (• x). The arrow at day 19 indicates the removal of the hypocotyl explant in all cultures, while at day 30 (second arrow) all cultures were transferred to fresh B5 medium with 2 gM 2,4-D
quired in order to produce large numbers of PEMs. Half of the medium in a standard starting culture was replaced by filter-sterilized, conditioned medium from a 7-d embryogenic suspension culture concomitant with removal of the hypocotyl explant at day 19. Further subculturing starting at day 30 was performed with fresh medium only. This treatment resulted in a dramatic increase in the number of proembryogenic masses observed and hence in embryogenic potential (Fig. 3). The levels attained were similar to levels normally observed at least I month later in untreated control cultures. Addition of 100- to 500-fold Amicon-concentrated conditioned medium, containing only material larger than 5 kDa produced the same promoting effect as unfractionated conditioned medium; heat treatment of this material destroyed the effect. Since conditioned medium devoid of the high-molecular-weight material did not give a promoting effect (Fig. 3), the possibility that phytohormones or other low-molecular-weight inducers excreted by the cells are the causative agents can
200
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
During the acquisition of embryogenic potential there is a gradual increase in the amount of a 72kDa extracellular protein that is also absent in media conditioned by somatic embryos. Taken together, the pattern of [35S]methionine-labeled extracellular polypeptides produced during the acquisition of embryogenic potential becomes increasingly complex in parallel to the increase in embryogenic potential.
Gene expression during development of embryogenic potential. The pattern of gene expression observed
Fig. 4. Extracellular polypeptides labeled with [35S]methionine at different times in a starting culture of carrot suspension cells and separated by 12.5% PAGE, For comparison, the extracellular polypeptides produced by an established high-density suspension culture and the embryos and its callus derived from such a culture are also shown
be excluded. None of the described culture treatments gave significant alterations of the macroscopic growth rates of the cultures when compared to the unsupplemented culture (data not shown).
Synthesis of extracellularpolypeptides during acquisition of embryogenic potential. Figure 4 shows the [35S] methionine-labeled proteins that are excreted into the medium of starting cultures at different times after inoculation. The extracellular-protein patterns of an established, high-density embryo~ genic culture and of somatic embryos produced from this culture are also shown. Before embryogenic potential is acquired (see Fig. 1 and 4, day 4), the pattern is surprisingly like that of an embryo culture, e.g. fairly high amounts of a 90-kDa protein. There appears to be a sudden increase at day 9 of a 27 kDa polypeptide which is present in embryogenic suspension cultures and is absent or greatly reduced during somatic embryogenesis.
during transition of the explant cells into an embryogenic suspension culture and in the somatic embryos generated from these cultures was analyzed by in-vitro translation of total cellular m R N A followed by two-dimensional PAGE analysis. Comparison of the patterns obtained from hypocotyl explants at day 14 (Fig. 5A), non-embryogenic suspension cells from a starting culture at day 14 (Fig. 5B), embryogenic suspension cells (Fig. 5C) and somatic embryos (Fig. 5D) show that the abundant m R N A populations visualized this way are quite similar. There are, however, a few notable differences observed in the translation products of the different developmental states examined. By day 14, callus formation at the cut surfaces of the hypocotyl explants is well underway. Comparison of in-vitrotranslation products from these explants and from suspension cells released from the explants at day 14 shows four spots are represented at lower levels in the released cell sample. Two products are present in the released cells that are not found in hypocotyl explant tissue (arrowheads in Fig. 5 A, B). At day 61, when embryogenic potential has developed in the starting culture, four other products (arrowheads in Fig. 5 C, open circles in Fig. 5A, B) have appeared that are not present in hypocotyl explants or in 14-d starting cultures. These in-vitro-translation products could represent genes involved in the acquisition of embryogenic potential. One product has disappeared from the pattern at day 61 (arrowheads in Fig. 5A, B and open circle in 5 C, D). Upon initiation of embryogenesis by dilution of proembryogenic masses in 2,4-D free medium, two products appear (arrowheads in Fig. 5 D, open circles in 5A, B, C) that are present only in somatic embryos; while six products disappear (arrowheads in Fig. 5A, B, C open circles in 5D). Three of the four in-vitrotranslation products that appeared first in 61-d embryogenic suspension cells increased considerably in somatic embryos (arrowheads in Fig. 5C, D, open circles in 5 A, B). One product is present only
S.C. de Vries et al. : Acquisitionof embryogenicpotential in carrot
201
Fig. 5A-D. Fluorographs of [35S]methionine-labeledin-vitro-translationproducts obtained from a carrot-mRNA-directed wheatgerm extract and separated by two-dimensionalPAGE as outlined in methods. A Hypocotylexplants after 14 d immersion in B5 mediumwith 2 ~tM2,4-D. B Non-embryogenicsuspension cells liberated from the explants at day 14. C Embryogenicsuspension cells at day 61 after initiation. D Somatic embryos. Arrowheads indicate in-vitro-translationproducts present in one pattern and absent in others (open circles)
in embryogenic cultures (arrowhead in Fig. 5C, open circles in 5A, B, C).
Expression of an embryo-regulated gene is associated with the acquisition of embrvogenic potential. Differential screening of a 2gtll embryo cDNA library with somatic embryo and suspension polyadenylated R N A has yielded several cDNA recombinants that on subsequent analysis showed a higher level of expression in somatic embryos than in proliferating cells of the embryogenic carrot line W D I . One of these cDNA sequences, designated Dc3, was highly expressed as m R N A in PEMs and somatic embryos (Thomas and Wilde 1985, 1987). The utility of Dc3 as a marker for the acquisition of embryogenic potential was further explored by
using it to probe R N A gel blots containing total RNA obtained from the Flakkese SG766 starting cell suspensions. The results of one experiment are shown in Fig. 6 and demonstrate that a 750-nucleotide transcript complementary to Dc3 is present in somatic embryos and to a much lower extent in unfractionated 51 d embryogenic starting cultures. Dc3 m R N A is undetectable in the non-embryogenic cells of 14-d and 22-d starting cultures. From this experiment, it is apparent that Dc3 is expressed only when PEMs are present in the starting cultures. Furthermore, these results clearly demonstrate that Dc3 expression is not a cellular response to culturing in the presence of 2,4-D. The pattern of expression demonstrated by Dc3 strongly indicates that proembryogenic masses, being an
202
Fig. 6. Patterns of RNA expression of an embryo-regulated cDNA, Dc3. Hybridization of Dc3 to a gel blot containing total RNA (8 gg per lane) extracted from carrot suspension cells at different times during acquisition of embryogenic potential. nt, nucleotides
essential prerequisite for somatic embryogenesis, can be considered as the first distinct and differentiated stage of somatic embryogenesis. Discussion
Somatic embryogenesis in Daucus carota has been studied extensively from a physiological point of view (Nomura and Komamine 1986), and although the molecular mechanisms that determine and control somatic embryogenesis are not yet understood, progress has been made in understanding the factors involved in the initiation and maintenance of this developmental pathway. Nomura and Komamine (1985) demonstrated that morphologically distinct single cells exist in suspension cultures maintained at high density and in the presence of high 2,4-D concentrations. These cells, designated Type 1 cells, are capable of developing into proembryogenic masses after exposure to low levels of 2,4-D. It is unclear at present whether these embryogenic cells derive from a common precursor cell type already present in the original explant or, alternatively, are continuously formed de novo from non-embryogenic cells. Subsequent events of the somatic embryogenesis pathway occur in the absence of exogenous 2,4-D and result in the gross morphogenetic changes leading to the formation of somatic embryos. In carrot, as well as in many other plant species, the synthetic auxin, 2,4-D, is the most efficient inducer of embryogenic potential (Ammirato 1983; Sung et al. 1984). This auxin analogue facilitates the development of somatic embryos up to the
S.C. de Vries et al. : Acquisition of embryogenic potential in carrot
globular stage, but it acts as a potent inhibitor of further development (Borkird et al. 1986). Virtually nothing is known about the molecular mechanisms by which 2,4-D induces embryogenic potential in plant tissue cultures. The experiments presented here demonstrate that carrot cells newly liberated from explant tissue have not yet acquired the capacity to produce somatic embryos. Although no attempt has been made to isolate the single cell precursors, or Type 1 cells, for proembryogenic masses during this period, it seems logical to assume that they are formed during the first two weeks of a starting culture. This period can be thought of as the time needed for cells to dedifferentiate and to obtain the competence to give rise to embryogenic cells, perhaps similar to the period in which Convolvulus leaf explants acquire the competence to produce shoots (Christianson and Warnick 1984, 1985). A stimulatory effect of conditioned medium on somatic embryogenesis has been demonstrated in carrot by Hari (1980) and Smith and Sung (1985). However, few attempts have been made to identify the responsible factors; although during regeneration of barley microspores, modified phytohormones have been found to be the active component (K6hler and Wenzel 1985). In contrast, our results indicate that the components of conditioned medium, active in the acquisition of embryogenic potential in starting cultures, are high-molecular-weight factors. This excludes the possibility that phytohormones, oligosaccharides (Tran Thanh Van et al. 1985), polyamines (Fienberg et al. 1984) or other low-molecular-weight components of conditioned medium are the causative agents. Because of the heat-lability of the effect, we tentatively conclude that excreted polypeptide(s) are the active components. Recently, Satoh et al. (1986) described a carrot extracellular glycoprotein of 65 kDa that appeared in the medium of somatic embryos only. In our analysis, however, we have not encountered a protein of identical molecular weight and behavior. A wide variety of lytic enzymes such as peroxidases (Chibbar et al. 1984), proteases and phosphatases (Wink 1984) are present in conditioned medium, but presently it is unknown whether any of these known excreted enzymes is involved in determination of embryogenic potential in carrot. It is noteworthy that a comparison of 3SS-labeled extracellular polypeptides produced during the acquisition of embryogenic potential shows a considerable increase in complexity parallel to the increasing embryogenic potential. The analysis of translational profiles of m R N A isolated from hypocotyl explants, starting suspen-
s.c. de Vries et al. : Acquisition of embryogenic potential in carrot sion cells, e m b r y o s a n d proliferating callus cells c o n f i r m s previous o b s e r v a t i o n s (Sung a n d O k i m o to 1981, 1983) t h a t differences in gene expression between o r g a n i z e d cells, like s o m a t i c e m b r y o s , a n d u n o r g a n i z e d cells, like callus a n d suspension cultures, are r e m a r k a b l y small a n d involve less t h a n ten i n - v i t r o - t r a n s l a t i o n p r o d u c t s out o f the a p p r o x imately 300 spots resolved on t w o - d i m e n s i o n a l gels between p H 4.5 a n d 7.5. The s a m e result was obtained if the t r a n s l a t i o n a l profiles o f the original explants a n d the derived suspension cells are c o m pared, indicating t h a t gross changes in the expression o f genes e n c o d i n g a b u n d a n t p o l y p e p t i d e s d o n o t o c c u r during dedifferentiation, induction o f embryogenic potential and formation of somatic e m b r y o s . M o s t differences were detected between s o m a t i c e m b r y o s a n d suspension cells f r o m starting cultures w i t h o u t p r o e m b r y o g e n i c masses. T h e gene c o r r e s p o n d i n g to the c D N A r e c o m binant, Dc3, is expressed in cultures t h a t c o n t a i n only p r o e m b r y o g e n i c masses or s o m a t i c e m b r y o s . This result, along with o b s e r v a t i o n s on the p a t terns o f gone expression in P E M s a n d s o m a t i c embryos, provides a m o l e c u l a r basis to the c o n c e p t t h a t specific genes involved in s o m a t i c e m b r y o g e n esis are already expressed long before m o r p h o l o g i cally discernible s o m a t i c e m b r y o s are present. T h e implications o f this finding are t h a t d e t e r m i n a t i o n o f cells destined to p r o d u c e s o m a t i c e m b r y o s takes place m u c h earlier t h a n previously a s s u m e d a n d u n d e r conditions t h a t inhibit the gross m o r p h o l o g ical changes associated with s o m a t i c e m b r y o g e n e sis. T h e availability o f the starting-culture system described here a n d m o l e c u l a r m a r k e r s such as Dc3 n o w allow the analysis o f the onset o f these e m b r y onic gene-expression p r o g r a m s . F u r t h e r m o r e , the starting cultures will be useful in the identification o f genes directly involved in the acquisition o f emb r y o g e n i c potential in c a r r o t suspension cultures. S o m e o f these genes are p r o b a b l y expressed u n d e r the influence o f 2,4-D a n d m a y encode extracellular proteins. We thank Piet Madern and Reindert de Fluiter for artwork and photography and Hedy Adriaansz, Rilla Eaton and Sandra Garcia for typing the manuscript. This work was supported in part by USDA Competitive Grants 84CRCR-I-1391 and 86CRCR-1-2143 awarded to T.L.T. Dayl:on Wilde is the recipient of a W.R. Grace fellowship.
References
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Note added in proof
Recently we have also demonstrated that extracellular proteins are involved in the development of somatic embryos from proembryogenic masses. (S.C. de Vries et al. Genes & Development 2, 462-476, 1988)
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Received 29 October/987; accepted 31 March 1988
