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Effect of culture densities on cell proliferation and regeneration from embryogenic cell suspensions of Picea sitchensis
Plant Science, 72 (1990) 115--123 Elsevier Scientific Publishers Ireland Ltd.
115
Effect of culture densities on cell proliferation and regeneration from embryogenic cell suspensions of Picea sitchensis P. Krogstrup Botanic Garden, University of Copenhagen, ~ster Farimagsgade 2B, DK-1353 Copenhagen K (Denmark) (Received March 30th, 1990; revision received June 6th, 1990; accepted June 26th, 1990)
Suspension cultures were established from embryogenic cell masses initiated from mature zygotic embryos of Sitka spruce (Picea sitchensis). The sedimented cell volume (SCV) of suspension cultures was used as a non-destructive parameter for quantifying the proliferation. The proliferation rate of the suspension culture was influenced by the relative culture density (ml SVC per 100 ml suspension) and the total volume of suspension culture per flask (at 2007o cell density). The differences in proliferation rate were ascribed an interaction between volume of culture medium and volume of culture atmosphere. An efficient system for controlled plating of the embryogenic suspension cultures is used to demonstrate the effect of plating density on regeneration (formation of "stage 2, 3 and 4 " somatic embryos (SE)). Plantlets, regenerated from suspension cultures, have been succesfully transferred to soil and exhibit normal growth and development as compared with seedlings.
Key words: Picea sitchensis; Sitka spruce; somatic embryogenesis; suspension culture; culture density; plating
Introduction
With the development of somatic embryogenic systems, for coniferous species, new perspectives have been opened for implementing biotechnological methods in forestry. In order to exploit the potential of somatic embryogenesis, it is necessary to develop reliable methods for suspension culture of the embryogenic cell masses. The genetic and embryogenic stability of coniferous embryogenic cells [1,2] combined with the potential for repetitive formation of proembryos [3] make these systems particularly suited for suspension culture. Use of suspension culture reduces the variation caused by gradients of chemical and physical factors and accelerates experimental response time as compared with solid medium culture which will ensure a more reliable experimental model system. Furthermore, suspension cultures are an excellent source for protoplast isolation and can be used in bioreactor technology and automation which are important for low cost plant multiplication [4].
In order to use embryogenic suspension culture as a reproducible tissue culture system it is imperative that one is able to quantify the parameters for growth and development. Embryogenic suspension cultures have so far been reported in Picea glauca [5--7], Picea abies [8], Pseudotsuga menziensii [9] and Pinus strobus [10]. However, these studies contain few quantitative data. In the present paper, a method for the establishment of a suspension culture of embryogenic cell masses of Sitka spruce (P. sitchensis) is presented with quantitative data on the effect of cell densities on proliferation of proembryos ("stage 1" somatic embryos (SE) [11]) and subsequent plant regeneration. Materials and Methods
Plant material Somatic embryogenic cell lines were obtained from mature seeds as previously described [12]. The embryogenic cell masses with proliferating "stage 1" embryos were subcultured weekly on
0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
116
modified BMI-S1 medium [13] with elevated levels of casein hydrolysate (acidic, Sigma Type I) (1000 ppm) and 2,4-D (10/aM), gelled with 0.18°70 Gelrite (Kelco).
Establishment and subculture of embryogenic suspension cultures Samples (100 mg, FW) of rapidly proliferating embryogenic cell masses were transferred to 250ml Erlenmeyer flasl~s with 25 ml liquid BMI-SI, pH 5.7 [13]. The flasks were sealed with Alu Caps (Struers, Copenhagen) and wrapped with two layers of 0.01 mm polyethylene film (Irma). Flasks were placed on a rotary shaker (100 rev./min, amplitude 1.5 cm.) in darkness at 24°C. Each week 20 ml BMI-S1 per flask were added to the cultures. When the culture volume reached I00 ml the cultures were poured into sterile, 100 ml measuring cylinders and allowed to sediment for 30 min. No initial cell debris was observed during the establishment. The resulting sedimented cell volume (SCV) was used routinely as a non-destructive quantitative measurement of growth or proliferation of "stage 1" SE in culture. The supernatant consisting of spent medium was discarded and new medium added in the ratio of 20:100 (ml SCV: ml total volume of suspension culture). Cell suspensions were subcultured weekly with this standard cell density. After 2--3 months a uniform and rapidly growing suspension culture consisting primarily of "stage 1" SE was established. It is important to stress that the SCV can be influenced by the turgidity of various cell types and should not be used as a quantitative measure in very heterogenous suspensions. However, in the present suspension cultures consisting primarily of "stage 1" SE, the SCV has shown to be a fairly accurate reflection of t h ( p r o l i f e r a t i o n of "stage 1" SE. In order to optimize the proliferation of "stage 1" embryos, various experiments were performed on established embryogenic suspension cultures. Prior to experiments, different batches (flasks) of suspension cultures from identical genotypes were pooled in order to reduce variation resulting from selection or physiological state. The SCV was used to quantify the proliferation. Suspension cultures were tested for microbial contaminations both
before and after experiments by plating on Difco Plate count agar.
Growth/proliferation curve for suspension culture Suspension cultures were cultivated as standard (density 20:100). The SCV was measured in each flask every other day for a period of 28 days. The measurement were repeated 6 times with one of the genotypes used in these experiments.
Experiment 1: Effect of culture density Different amounts of embryogenic cells were cultured in 250 ml erlenmeyer flasks as above, resulting in the following initial suspension culture densities (3.8:100; 7.5:100; 15.0:100; 30.0:100; 60:100 ( ml SCV: total ml of suspension culture). The SCV was measured weekly during four subcultures with the above culture densities. Experiments were repeated three times with new suspension culture material. Data from two different genotypes were pooled.
Experiment 2: Effect of total volume of a (20:100) suspension culture per flask Different amounts (6.3, 12.5, 25, 50, 100 and 200 ml) of a (20:100) suspension culture were cultured in 250-ml flasks resulting in the following amounts of embryogenic cell masses per flask: 1.25, 2.5, 5.0, 10.0, 20.0, 40.0 ml SCV. The SCV was measured weekly during four subcultures with the above amounts of suspension culture. Experiments were repeated three times with new suspension culture material. Data from two genotypes were pooled. Suspension cultures from experiments 1 and 2 were transferred back to standard culture conditions in order to test reversibility of the observed growth responses. Additionally, cultures were observed microscopically after experiments in order to observe qualitative differences. The available media and void volume (culture atmosphere) for the different treatments in experiment 1 and 2 are shown in Fig. 1.
Regeneration of plantlets: in vitro culture media for regeneration. For the first 3 weeks, the plated "stage 1" SE were cultured on a BMG-1 medium[13] modified with 550 laM inositol, 2/aM
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thiamin--HCl, 100 ppm casein hydrolysate, 88 mM sucrose and supplied with 5/aM ABA (Sigma A 1049 _ cis-trans isomer) for maturation [12]. After 3 weeks, the maturation medium was substituted with the same medium without ABA. After 4 weeks, the medium was substituted with BMG-2 [12] without growth regulators or casein hydrolysate. For the rest of the in vitro culture period, the cultures were subcultured with 4-week intervals on this medium.
Method for quantitative plating of suspension cultures. In order to quantify the regeneration processes, a plating system was developed. A defined amount of SCV cultured under standard conditions was suspended in maturation medium
without ABA to a t o t a l volume of 25 ml. This suspension was then poured in a BiJchner funnel with a glass filter (diameter 60 mm, porosity 40--100 ~ n ) onto which a sterile filter paper (diameter 60 mm) had been placed. The filter funnel was placed on a Biichner filter flask. By applying a light vacuum, the suspended cells sedimented uniformly on the filter paper disc. The filter paper with the defined amount of cells was subsequently transferred to the culture system with sterile forceps. The filter papers used were cut from sheets of filter paper used for making tea bags (Irma, Copenhagen). In vitro culture system. The culture system used for regeneration consisted of a 60 x 20 mm disc of non-woven polyester fleece (Fredensborg Indk0bscentral, Copenhagen) placed in a 370-ml jar and soaked with approx. 70 ml of liquid medium. The filter paper with the sedimented cells were placed on top of the polyester fleece. Jars were closed with transparent plastic screw-on lids and sealed with two rounds of 0.01 mm polyethylene film. Media were changed by removing the spent medium with a pipette and adding fresh.
Experiment 3: Effect of plating density on regeneration In order to test the effect of cell densities on the regeneration processes, different amounts (0.62, 1.25, 2.5, 5.0 and 10 ml SCV per filter disc) of cell suspension were plated as mentioned above. The number of "stage 2, 3 and 4 " SE [11] were counted at intervals of 3, 7 and 11 weeks after plating. The classification of "stage 4 " was based on the morphology of the top, i.e., early "stage 4 " with or without radicles. 1 ml SCV of the suspensions used contained approx. 400--500 "stage 1" SE and weighed approx. 0.2 g. Experiments were repeated 16 times with a total of 2 genotypes during the period of May 12--July 6. Statistics. Data were analyzed by single sided analysis of variance.
Conversion of somatic embryo Early "stage 4 " SE were removed from the filter paper and placed on a new filter paper and fleece soaked with fresh medium at density of 30 - 10 per jar.
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Physical milieu Somatic embryos on maturation medium were kept at 24°C -+ 1 °C in darkness. After maturation for 3 weeks the cultures were transferred to a 16:8-h photoperiod at 24°C _+ 1 °C. Light sources used were Philips T L D 15 W/33 lamps at an intensity of 10--30/xE m -2 s-l at 24°C. Reestablishment o f plantlets ex vitrum Somatic embryos with emerging roots (2--5 mm) were transferred to an autoclaved mixture of peat/perlite/vermiculite (1:1:1) in styrofoam boxes (27 X 20 x 7 cm) in a greenhouse at 22°C ± 5°C at 90% RH (Airfog ultrasound nozzles). After 6 weeks the plantlets were transferred to ambient R H at 20°C ± 5°C. Some of the plantlets were cultivated under accelerated growth conditions by long photoperiod.
density on the proliferation in embryogenic cell suspensions was observed in experiment 1 (Fig. 3A). In experiment 2 the available volume of medium and void volume are different from experiment l (Fig. 1). However, a significant (P < 0.1%) effect of culture density on the additional proliferation is still noted (Fig. 3A). Furthermore, the optimal culture density is approx. 10 ml SCV per flask in both experiments, although the levels of additional proliferation differ. In Fig. 3B the additional proliferation per ml SCV are plotted for the different culture densities and shows a decline in proliferation with increasing culture density. This is noted in both experiments 1 and 2.
Results 25
Growth proliferation curve f o r the suspension cultures In Fig. 2. the growth or proliferation in suspension culture is plotted for one genotype. Apart from an insignificant lag phase, the cultures show a linear growth until day 26--28 where the growth diminishes and the cultures eventually deteriorate.
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Fig. 3. A: additional growth or proliferation per culture flask after 7 days of culture as a function of culture density (ml SCV per culture flask). B: additional growth/proliferation per ml SCV as a function of cell density. LSD values (95°70) represented for means of 12 replicates with 2 genotypes.
119
for the different plating densities. A significant (P < 5070) effect of plating density on formation of "stage 2" SE (Figs. 5C and 5D) is seen 3 weeks after plating (Fig. 4). The increase in regeneration at higher plating densities is also significant (P < 1%) for "stage 3" SE (Fig. 5E) after 7 weeks and for "stage 4" SE (P < 5°70) (Fig. 5F) after 11 weeks. As for the number of somatic embryos per ml plated SCV there is a significantly (P < 1070) higher number of "stage 3" at lower plating densi-
The re-growth test under standard culture conditions demonstrated reversibility of the observed differences in proliferation rate. A microscopic evaluation of the suspensions did not show any obvious difference in the proportion of "stage 1" somatic embryos (Figs. 5A and 5B) relative to single cells and cell clusters.
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Fig. 4. Number of different stages of somatic embryos at different plating densities counted 3, 7 and 11 weeks after plating. 0.5 × LSD values (95 %) represented for means of 16 replicates with 2 genotypes.
Fig. 5. A: Somatic embryogenic suspension with "stage 1" SE (somatic embryo). B: Close up of "stage 1" SE. C: Plated "stage 2" SE, 3 weeks after plating. D: Close up of "stage 2" SE. E: Plated "stage 3" SE, 7 weeks after plating. F: Planted "stage 4" SE, 14 weeks after plating. G: Reestablished plants regenerated from suspension cultured "stage l " SE, approx. 8 months after plating. H: Reestablished plants approx. 20 months after plating.
121 ties after 3 weeks. The same significant tendency is noted for "stage 4" ( P < l°70) after 7 weeks and is almost significant after 11 weeks.
Conversion o f somatic embryos and reestablishment ex vitrum Approximately 50°7o of the early "stage 4" embryos demonstrated radicle emergence and development. Ninety per cent of the embryos with roots survived the explantation ex vitrum and demonstrated normal growth and development (Figs. 5G and 5H). Discussion
Suspension cultures normally exhibit a typical sigmoid growth curve with a lag phase followed by an exponential-, linear-, deceleration- and stationary growth phase [14]. In the present suspension culture system, there is only a slight lag-phase followed by a linear growth phase and ending with a stationary phase. In this context it is important to emphasize that the present embryogenic suspension cultures primarily consist of suspended organized structures ("stage 1" SE) with a minor fraction of single cells and cell clusters. Somatic embryogenesis in conifers is considered to represent the PEDC (pre-embryogenic-determined cells) pattern of somatic embryogenesis [3]. In these systems "stage 1" SE will form by some sort of cleavage processes from existing "stage 1" SE, resulting in repetitive somatic embryogenesis. This pattern of formation could explain the insignificant lag phase representing a minor need for conditioning of the culture medium which is also reflected in the responses to different culture densities (experiment 1 and 2, Fig. 3B).
Effect o f culture densities The reversibility of the observed differences in the rate of proliferation, indicates that the experimental responses were caused by the imposed culture conditions rather than selection of cell lines with different proliferation potential. Several causal factors for the observed differences in proliferation rate in relation to culture densities can be suggested. The proliferation curve in experiment 1 (Fig. 3A) could be explained by a
need for conditioning factors at low culture density and exhaustion of medium constituents at high culture density. In Fig. 3B the proliferation rate per ml SCV is seen to correlate with availability of medium per ml SCV (Fig. 1). In experiment 2 there is an overall lower proliferation rate which could be explained by the lower medium availability (up to culture density of 20 ml SCV) as compared with experiment 1. However, the decline in proliferation rate at culture densities higher than 10 ml SCV cannot solely be explained in terms of medium availability. This is also seen in Fig. 3B where the amount of available medium per ml SCV is constant in the tested range of culture densities (Fig. 1) and shows no correlation with the observed decline in proliferation. Another factor which may be considered is the gaseous atmosphere in the culture flasks. The volume of culture atmosphere has been shown to modulate differentiation and growth processes in vitro [15,16]. In vitro plant tissue cultures produce a number of gaseous or volatile compounds [17,18]. Some of these compounds which have been shown to affect growth and differentiation processes in vitro include ethylene [19--22], oxygen [23], carbondioxide [22,24--26] and ethanol I27]. The importance of the volume of the culture atmosphere is indicated in Fig. 3B where the proliferation rate per ml SCV is seen to correlate partially with the volume of culture atmosphere per ml SCV in both experiments (Fig. 1). In experiment 2 the different culture volumes also result in different degrees of agitation and culture surface area which should also be considered.
Effect o f plating densities on regeneration Plating of suspension cultures normally requires an optimal cell density. At suboptimal densities, cells are not able to condition the medium for sustained growth. On the other hand supraoptimal densities result in competition for available growth factors In experiment 3 the optimal plating density for "stage 3" and "stage 4" SE is between 2.5 and 5 ml SCV. In the tested range of SCV the lower regeneration at lower culture densities is primarily caused by a reduced number of initial propagules
122
("stage 1" SE) rather than a need for conditioning factors. This is seen by the high plating efficiency (number of SE per ml SCV) at low culture densities. This can be ascribed to the fact that the regeneration during plating is merely a continued development of "stage 1" SE formed in the suspension phase.
Conclusion By the use of suspension culture, proembryos ("stage 1") can be produced continuously. In the present suspension cultures consisting primarily of "stage 1" SE, the proliferation can routinely and non-destructively be quantified by use of the SCV. This should ideally be coupled with some qualitative evaluation of the relative fractions of the culture by microscopic counting or fractionation by gradient centrifugation. The ability to produce large numbers of somatic embryos of distinct developmental stages in suspension ("stage 1") and by plating ("stage 2, 3 and 4") makes the system beneficial as a model system for quantitative embryological studies. Similarly the possibility to produce large amounts of plantlets makes the system interesting for production purposes. The multiplication phase can easily be performed in suspension culture and use of polyester fleece as tissue carrier makes it easy to change media without disturbing the cultures.
Acknowledgements Thanks to Minna Bendixen, Dorthe H. Christiansen, Leif Jensen and Cherry Nielsen for excellent technical assistance, Cherry Nielsen also for linguistic revision and to Jens V. NOrgaard for review of the manuscript. The work was financed by Danish Agricultural and Veterinary Research Council Grant No. 13-4104 and the Velux foundation of 1981.
References L.H. Mo, S. von Arnold and U. Lagercranz, Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies L.Karst). Plant Cell Rep., 8 (1989) 375--378.
2
A. Schuller, G. Reuter and T. Geier, Somatic embryogenesis from seed explants of Abies alba. Plant Cell Tiss. Org. Cult., 17 (1989) 53--58. 3 S.R. Wann, Somatic embryogenesis in woody species. Hortic. Rev., 10 (1988) 153--181. 4 M.R. Becwar, S.R. Wann, M.A. Johnson, S.A. Vethagen, R.P. Feirer and R. Nagmani, Development and characterization of in vitro embryogenic systems in conifers, in: M.R. Ahuja (Ed.), Somatic Cell Genetics of Woody Plants, Kluwer Academic Publishers, 1988, pp. 1 --18. 5 S.M. Attree, D.I Dunstan and L.C. Fowke, Initiation of embryogenic callus and suspension cultures and improved embryo regeneration from protoplasts, of white spruce (Picea glauca). Can. J. Bot., 67 (1989) 1790--1795. 6 I. Hakman and S. von Arnold, Somatic embryogenesis and plant regeneration from suspension cultures of Picea glauca (White spruce). Physiol. Plant., 72 0988) 579-587. 7 I. Hakman and L.C. Fowke, An embryogenic cell suspension culture of Picea glauca (White spruce). Plant Cell Rep., 6 (1987) 20--22. 8 M.P. Boulay, P.K. Gupta, P. Krogstrup and D.J. Durzan, Development of somatic embryos from cell suspension cultures of Norway spruce (Picea abies Karst.). Plant Cell Rep., 7 (1988) 134--137. 9 D.J. Durzan and P.K. Gupta, Somatic embryogenesis and polyembryogenesis in Douglas-fir cell suspension cultures. Plant Sci., 52 (1987) 229--235. 10 J.J. Finer, H.B. Kriebel and M.R. Becwar, Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.). Plant Cell Rep., 8 (1989) 203--206. 11 S. von Arnold and I. Hakman, Regulation of somatic embryo development in Picea abies by abscisic acid (ABA). J. Plant Physiol., 132 0988) 164---169. 12 P. Krogstrup, E.N. Eriksen, J.D. Moller and H. Roulund, Somatic embryogenesis in Sitka spruce (Picea sitchens/s (Bong.) (Carr.). Plant Cell Rep., 7 (1988) 594-597. 13 P. Krogstrup, Embryolike structures from cotyledons and ripe embryos of Norway spruce (Picea abies). Can. J. For. Res., 16 (1986) 664--668. 14 H.E. Street, Cell (suspension) cultures-techniques, in: H. Street (Ed.), Plant Tissue and Cell Culture, Botanical Monographs, Vol. l l , Blackwell Scientific Publications, 1973, pp. 59--99. 15 S.J. Dalton and P.J. Dale, Induced tillering of Lolium multiflorum in vitro. Plant Cell Tiss. Org. Cult., 1 (1981) 57--64. 16 J.M. Dunwell, In vitro regeneration from excised leaf discs of three Brassica species. J. Exp. Bot., 32 (1981) 789 --799. 17 D.Thomas and T. Murashige, Volatile emissions of plant tissue cultures. I. Identification of the major components. In Vitro, 15 (1979) 654--658. 18 I.K. Heitzmann and W. Schultze, Fliichtige inhaltstoffe
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19
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in pflanzlichen cellkulturen-eine iibersicht. Biochem. Physiol. Pflanzen., 184 (1989) 3--30. T.J. Huxter, D.M. Reid and T.A. Thorpe, Ethylene production by tobacco (Nicotiana tabacum) callus. Physiol. Plant., 46 (1979) 374--380. T.J. Huxter, T.A. Thorpe and D.H. Reid, Shoot initiation in light- and dark-grown tobacco callus: the role of ethylene. Physiol. Plant., 53 (1981) 319--326. P.P. Kumar, D.M. Reid and T.A. Thorpe, The role of ethylene and carbon dioxide in differentiation of shoot buds in excised cotyledons of Pinus radiata in vitro. Physiol. Plant., 69 (1987) 244--252. P.P. Kumar, R.W. Joy IV and T.A. Thorpe, Ethylene and carbondioxide accumulation and growth of cell suspension cultures of Picea glauca (White spruce). J. Plant Physiol., 135 (1989) 592--596. R.H.J. Kessel and A.H. Carr, The effect of dissolved oxygen concentration on growth and differentiation of
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carrot (Daucus carota) tissue. J. Exp. Bot., 23 (1972) 996 - - 1007. J.P. Ducos, G. Feron and A. Pareilleux, Growth and activities of enzymes of primary metabolism in batch cultures under different pCO 2 conditions. Plant Cell Tiss. Org. Cult., 13 (1988) 167--177. P.K. Hegarty, N.J. Smart, A.H. Scragg and M.W. Fowler, The aeration of Cataranthus roseus L.G.Don suspension cultures in airlift bioreactors: The inhibitory effect of high aeration rates on culture growth. J. Exp. Bot., 37 (1986) 1911--1920. R.W. Zobel, Gaseous compounds of soybean tissue cultures: carbon dioxide and ethylene evolution. Environ. Exp. Bot., 27 (1987) 223--226. P. Perata, A. Alpi and F. LoSchiavo, Influence of ethanol on plant ceils and tissues. J. Plant Physiol., 126 (1986) 181--188.
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Effect of culture densities on cell proliferation and regeneration from embryogenic cell suspensions of Picea sitchensis P. Krogstrup Botanic Garden, University of Copenhagen, ~ster Farimagsgade 2B, DK-1353 Copenhagen K (Denmark) (Received March 30th, 1990; revision received June 6th, 1990; accepted June 26th, 1990)
Suspension cultures were established from embryogenic cell masses initiated from mature zygotic embryos of Sitka spruce (Picea sitchensis). The sedimented cell volume (SCV) of suspension cultures was used as a non-destructive parameter for quantifying the proliferation. The proliferation rate of the suspension culture was influenced by the relative culture density (ml SVC per 100 ml suspension) and the total volume of suspension culture per flask (at 2007o cell density). The differences in proliferation rate were ascribed an interaction between volume of culture medium and volume of culture atmosphere. An efficient system for controlled plating of the embryogenic suspension cultures is used to demonstrate the effect of plating density on regeneration (formation of "stage 2, 3 and 4 " somatic embryos (SE)). Plantlets, regenerated from suspension cultures, have been succesfully transferred to soil and exhibit normal growth and development as compared with seedlings.
Key words: Picea sitchensis; Sitka spruce; somatic embryogenesis; suspension culture; culture density; plating
Introduction
With the development of somatic embryogenic systems, for coniferous species, new perspectives have been opened for implementing biotechnological methods in forestry. In order to exploit the potential of somatic embryogenesis, it is necessary to develop reliable methods for suspension culture of the embryogenic cell masses. The genetic and embryogenic stability of coniferous embryogenic cells [1,2] combined with the potential for repetitive formation of proembryos [3] make these systems particularly suited for suspension culture. Use of suspension culture reduces the variation caused by gradients of chemical and physical factors and accelerates experimental response time as compared with solid medium culture which will ensure a more reliable experimental model system. Furthermore, suspension cultures are an excellent source for protoplast isolation and can be used in bioreactor technology and automation which are important for low cost plant multiplication [4].
In order to use embryogenic suspension culture as a reproducible tissue culture system it is imperative that one is able to quantify the parameters for growth and development. Embryogenic suspension cultures have so far been reported in Picea glauca [5--7], Picea abies [8], Pseudotsuga menziensii [9] and Pinus strobus [10]. However, these studies contain few quantitative data. In the present paper, a method for the establishment of a suspension culture of embryogenic cell masses of Sitka spruce (P. sitchensis) is presented with quantitative data on the effect of cell densities on proliferation of proembryos ("stage 1" somatic embryos (SE) [11]) and subsequent plant regeneration. Materials and Methods
Plant material Somatic embryogenic cell lines were obtained from mature seeds as previously described [12]. The embryogenic cell masses with proliferating "stage 1" embryos were subcultured weekly on
0168-9452/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
116
modified BMI-S1 medium [13] with elevated levels of casein hydrolysate (acidic, Sigma Type I) (1000 ppm) and 2,4-D (10/aM), gelled with 0.18°70 Gelrite (Kelco).
Establishment and subculture of embryogenic suspension cultures Samples (100 mg, FW) of rapidly proliferating embryogenic cell masses were transferred to 250ml Erlenmeyer flasl~s with 25 ml liquid BMI-SI, pH 5.7 [13]. The flasks were sealed with Alu Caps (Struers, Copenhagen) and wrapped with two layers of 0.01 mm polyethylene film (Irma). Flasks were placed on a rotary shaker (100 rev./min, amplitude 1.5 cm.) in darkness at 24°C. Each week 20 ml BMI-S1 per flask were added to the cultures. When the culture volume reached I00 ml the cultures were poured into sterile, 100 ml measuring cylinders and allowed to sediment for 30 min. No initial cell debris was observed during the establishment. The resulting sedimented cell volume (SCV) was used routinely as a non-destructive quantitative measurement of growth or proliferation of "stage 1" SE in culture. The supernatant consisting of spent medium was discarded and new medium added in the ratio of 20:100 (ml SCV: ml total volume of suspension culture). Cell suspensions were subcultured weekly with this standard cell density. After 2--3 months a uniform and rapidly growing suspension culture consisting primarily of "stage 1" SE was established. It is important to stress that the SCV can be influenced by the turgidity of various cell types and should not be used as a quantitative measure in very heterogenous suspensions. However, in the present suspension cultures consisting primarily of "stage 1" SE, the SCV has shown to be a fairly accurate reflection of t h ( p r o l i f e r a t i o n of "stage 1" SE. In order to optimize the proliferation of "stage 1" embryos, various experiments were performed on established embryogenic suspension cultures. Prior to experiments, different batches (flasks) of suspension cultures from identical genotypes were pooled in order to reduce variation resulting from selection or physiological state. The SCV was used to quantify the proliferation. Suspension cultures were tested for microbial contaminations both
before and after experiments by plating on Difco Plate count agar.
Growth/proliferation curve for suspension culture Suspension cultures were cultivated as standard (density 20:100). The SCV was measured in each flask every other day for a period of 28 days. The measurement were repeated 6 times with one of the genotypes used in these experiments.
Experiment 1: Effect of culture density Different amounts of embryogenic cells were cultured in 250 ml erlenmeyer flasks as above, resulting in the following initial suspension culture densities (3.8:100; 7.5:100; 15.0:100; 30.0:100; 60:100 ( ml SCV: total ml of suspension culture). The SCV was measured weekly during four subcultures with the above culture densities. Experiments were repeated three times with new suspension culture material. Data from two different genotypes were pooled.
Experiment 2: Effect of total volume of a (20:100) suspension culture per flask Different amounts (6.3, 12.5, 25, 50, 100 and 200 ml) of a (20:100) suspension culture were cultured in 250-ml flasks resulting in the following amounts of embryogenic cell masses per flask: 1.25, 2.5, 5.0, 10.0, 20.0, 40.0 ml SCV. The SCV was measured weekly during four subcultures with the above amounts of suspension culture. Experiments were repeated three times with new suspension culture material. Data from two genotypes were pooled. Suspension cultures from experiments 1 and 2 were transferred back to standard culture conditions in order to test reversibility of the observed growth responses. Additionally, cultures were observed microscopically after experiments in order to observe qualitative differences. The available media and void volume (culture atmosphere) for the different treatments in experiment 1 and 2 are shown in Fig. 1.
Regeneration of plantlets: in vitro culture media for regeneration. For the first 3 weeks, the plated "stage 1" SE were cultured on a BMG-1 medium[13] modified with 550 laM inositol, 2/aM
117 E-~
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thiamin--HCl, 100 ppm casein hydrolysate, 88 mM sucrose and supplied with 5/aM ABA (Sigma A 1049 _ cis-trans isomer) for maturation [12]. After 3 weeks, the maturation medium was substituted with the same medium without ABA. After 4 weeks, the medium was substituted with BMG-2 [12] without growth regulators or casein hydrolysate. For the rest of the in vitro culture period, the cultures were subcultured with 4-week intervals on this medium.
Method for quantitative plating of suspension cultures. In order to quantify the regeneration processes, a plating system was developed. A defined amount of SCV cultured under standard conditions was suspended in maturation medium
without ABA to a t o t a l volume of 25 ml. This suspension was then poured in a BiJchner funnel with a glass filter (diameter 60 mm, porosity 40--100 ~ n ) onto which a sterile filter paper (diameter 60 mm) had been placed. The filter funnel was placed on a Biichner filter flask. By applying a light vacuum, the suspended cells sedimented uniformly on the filter paper disc. The filter paper with the defined amount of cells was subsequently transferred to the culture system with sterile forceps. The filter papers used were cut from sheets of filter paper used for making tea bags (Irma, Copenhagen). In vitro culture system. The culture system used for regeneration consisted of a 60 x 20 mm disc of non-woven polyester fleece (Fredensborg Indk0bscentral, Copenhagen) placed in a 370-ml jar and soaked with approx. 70 ml of liquid medium. The filter paper with the sedimented cells were placed on top of the polyester fleece. Jars were closed with transparent plastic screw-on lids and sealed with two rounds of 0.01 mm polyethylene film. Media were changed by removing the spent medium with a pipette and adding fresh.
Experiment 3: Effect of plating density on regeneration In order to test the effect of cell densities on the regeneration processes, different amounts (0.62, 1.25, 2.5, 5.0 and 10 ml SCV per filter disc) of cell suspension were plated as mentioned above. The number of "stage 2, 3 and 4 " SE [11] were counted at intervals of 3, 7 and 11 weeks after plating. The classification of "stage 4 " was based on the morphology of the top, i.e., early "stage 4 " with or without radicles. 1 ml SCV of the suspensions used contained approx. 400--500 "stage 1" SE and weighed approx. 0.2 g. Experiments were repeated 16 times with a total of 2 genotypes during the period of May 12--July 6. Statistics. Data were analyzed by single sided analysis of variance.
Conversion of somatic embryo Early "stage 4 " SE were removed from the filter paper and placed on a new filter paper and fleece soaked with fresh medium at density of 30 - 10 per jar.
118
Physical milieu Somatic embryos on maturation medium were kept at 24°C -+ 1 °C in darkness. After maturation for 3 weeks the cultures were transferred to a 16:8-h photoperiod at 24°C _+ 1 °C. Light sources used were Philips T L D 15 W/33 lamps at an intensity of 10--30/xE m -2 s-l at 24°C. Reestablishment o f plantlets ex vitrum Somatic embryos with emerging roots (2--5 mm) were transferred to an autoclaved mixture of peat/perlite/vermiculite (1:1:1) in styrofoam boxes (27 X 20 x 7 cm) in a greenhouse at 22°C ± 5°C at 90% RH (Airfog ultrasound nozzles). After 6 weeks the plantlets were transferred to ambient R H at 20°C ± 5°C. Some of the plantlets were cultivated under accelerated growth conditions by long photoperiod.
density on the proliferation in embryogenic cell suspensions was observed in experiment 1 (Fig. 3A). In experiment 2 the available volume of medium and void volume are different from experiment l (Fig. 1). However, a significant (P < 0.1%) effect of culture density on the additional proliferation is still noted (Fig. 3A). Furthermore, the optimal culture density is approx. 10 ml SCV per flask in both experiments, although the levels of additional proliferation differ. In Fig. 3B the additional proliferation per ml SCV are plotted for the different culture densities and shows a decline in proliferation with increasing culture density. This is noted in both experiments 1 and 2.
Results 25
Growth proliferation curve f o r the suspension cultures In Fig. 2. the growth or proliferation in suspension culture is plotted for one genotype. Apart from an insignificant lag phase, the cultures show a linear growth until day 26--28 where the growth diminishes and the cultures eventually deteriorate.
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Fig. 3. A: additional growth or proliferation per culture flask after 7 days of culture as a function of culture density (ml SCV per culture flask). B: additional growth/proliferation per ml SCV as a function of cell density. LSD values (95°70) represented for means of 12 replicates with 2 genotypes.
119
for the different plating densities. A significant (P < 5070) effect of plating density on formation of "stage 2" SE (Figs. 5C and 5D) is seen 3 weeks after plating (Fig. 4). The increase in regeneration at higher plating densities is also significant (P < 1%) for "stage 3" SE (Fig. 5E) after 7 weeks and for "stage 4" SE (P < 5°70) (Fig. 5F) after 11 weeks. As for the number of somatic embryos per ml plated SCV there is a significantly (P < 1070) higher number of "stage 3" at lower plating densi-
The re-growth test under standard culture conditions demonstrated reversibility of the observed differences in proliferation rate. A microscopic evaluation of the suspensions did not show any obvious difference in the proportion of "stage 1" somatic embryos (Figs. 5A and 5B) relative to single cells and cell clusters.
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In Fig. 4, the regeneration profiles are shown
No. somatic embryos (SE) per plating. Week 3.
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Fig. 4. Number of different stages of somatic embryos at different plating densities counted 3, 7 and 11 weeks after plating. 0.5 × LSD values (95 %) represented for means of 16 replicates with 2 genotypes.
Fig. 5. A: Somatic embryogenic suspension with "stage 1" SE (somatic embryo). B: Close up of "stage 1" SE. C: Plated "stage 2" SE, 3 weeks after plating. D: Close up of "stage 2" SE. E: Plated "stage 3" SE, 7 weeks after plating. F: Planted "stage 4" SE, 14 weeks after plating. G: Reestablished plants regenerated from suspension cultured "stage l " SE, approx. 8 months after plating. H: Reestablished plants approx. 20 months after plating.
121 ties after 3 weeks. The same significant tendency is noted for "stage 4" ( P < l°70) after 7 weeks and is almost significant after 11 weeks.
Conversion o f somatic embryos and reestablishment ex vitrum Approximately 50°7o of the early "stage 4" embryos demonstrated radicle emergence and development. Ninety per cent of the embryos with roots survived the explantation ex vitrum and demonstrated normal growth and development (Figs. 5G and 5H). Discussion
Suspension cultures normally exhibit a typical sigmoid growth curve with a lag phase followed by an exponential-, linear-, deceleration- and stationary growth phase [14]. In the present suspension culture system, there is only a slight lag-phase followed by a linear growth phase and ending with a stationary phase. In this context it is important to emphasize that the present embryogenic suspension cultures primarily consist of suspended organized structures ("stage 1" SE) with a minor fraction of single cells and cell clusters. Somatic embryogenesis in conifers is considered to represent the PEDC (pre-embryogenic-determined cells) pattern of somatic embryogenesis [3]. In these systems "stage 1" SE will form by some sort of cleavage processes from existing "stage 1" SE, resulting in repetitive somatic embryogenesis. This pattern of formation could explain the insignificant lag phase representing a minor need for conditioning of the culture medium which is also reflected in the responses to different culture densities (experiment 1 and 2, Fig. 3B).
Effect o f culture densities The reversibility of the observed differences in the rate of proliferation, indicates that the experimental responses were caused by the imposed culture conditions rather than selection of cell lines with different proliferation potential. Several causal factors for the observed differences in proliferation rate in relation to culture densities can be suggested. The proliferation curve in experiment 1 (Fig. 3A) could be explained by a
need for conditioning factors at low culture density and exhaustion of medium constituents at high culture density. In Fig. 3B the proliferation rate per ml SCV is seen to correlate with availability of medium per ml SCV (Fig. 1). In experiment 2 there is an overall lower proliferation rate which could be explained by the lower medium availability (up to culture density of 20 ml SCV) as compared with experiment 1. However, the decline in proliferation rate at culture densities higher than 10 ml SCV cannot solely be explained in terms of medium availability. This is also seen in Fig. 3B where the amount of available medium per ml SCV is constant in the tested range of culture densities (Fig. 1) and shows no correlation with the observed decline in proliferation. Another factor which may be considered is the gaseous atmosphere in the culture flasks. The volume of culture atmosphere has been shown to modulate differentiation and growth processes in vitro [15,16]. In vitro plant tissue cultures produce a number of gaseous or volatile compounds [17,18]. Some of these compounds which have been shown to affect growth and differentiation processes in vitro include ethylene [19--22], oxygen [23], carbondioxide [22,24--26] and ethanol I27]. The importance of the volume of the culture atmosphere is indicated in Fig. 3B where the proliferation rate per ml SCV is seen to correlate partially with the volume of culture atmosphere per ml SCV in both experiments (Fig. 1). In experiment 2 the different culture volumes also result in different degrees of agitation and culture surface area which should also be considered.
Effect o f plating densities on regeneration Plating of suspension cultures normally requires an optimal cell density. At suboptimal densities, cells are not able to condition the medium for sustained growth. On the other hand supraoptimal densities result in competition for available growth factors In experiment 3 the optimal plating density for "stage 3" and "stage 4" SE is between 2.5 and 5 ml SCV. In the tested range of SCV the lower regeneration at lower culture densities is primarily caused by a reduced number of initial propagules
122
("stage 1" SE) rather than a need for conditioning factors. This is seen by the high plating efficiency (number of SE per ml SCV) at low culture densities. This can be ascribed to the fact that the regeneration during plating is merely a continued development of "stage 1" SE formed in the suspension phase.
Conclusion By the use of suspension culture, proembryos ("stage 1") can be produced continuously. In the present suspension cultures consisting primarily of "stage 1" SE, the proliferation can routinely and non-destructively be quantified by use of the SCV. This should ideally be coupled with some qualitative evaluation of the relative fractions of the culture by microscopic counting or fractionation by gradient centrifugation. The ability to produce large numbers of somatic embryos of distinct developmental stages in suspension ("stage 1") and by plating ("stage 2, 3 and 4") makes the system beneficial as a model system for quantitative embryological studies. Similarly the possibility to produce large amounts of plantlets makes the system interesting for production purposes. The multiplication phase can easily be performed in suspension culture and use of polyester fleece as tissue carrier makes it easy to change media without disturbing the cultures.
Acknowledgements Thanks to Minna Bendixen, Dorthe H. Christiansen, Leif Jensen and Cherry Nielsen for excellent technical assistance, Cherry Nielsen also for linguistic revision and to Jens V. NOrgaard for review of the manuscript. The work was financed by Danish Agricultural and Veterinary Research Council Grant No. 13-4104 and the Velux foundation of 1981.
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2
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