Appl Microbiol Biotechnol (1992) 38:46-51

App//ed Microbiology Biotechnology © Springer-Verlag 1992

Bioreactor culture of Picea mariana Mill. (black spruce) and the species complex Picea glauca-engelmannii (interior spruce) somatic embryos. Growth parameters* T. E. Tautorus, M. M. Lulsdorf, S. I. Kikcio, and D. I. Dunstan Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9 Received 11 March 1992/Accepted 12 June 1992

Summary. Somatic embryo cultures of Picea mariana and the species complex P. glauca-engelmannii were each grown in 7.5-1-capacity mechanically-stirred bioreactors containing 6 1 medium (LP, von Arnold and Eriksson) with 30 mM sucrose. Growth of both species occurred with no observable signs of shear stress due to mechanical agitation. Growth kinetics were analysed using an array of parameters (settled culture volume, packed culture volume, osmolarity, conductivity, pH). These were compared with flesh weight, dry weight, and somatic embryo number in order to determine what parameters were highly correlated with growth and embryo number. Increasing the sucrose concentration from 30 mM to 60 mM resulted in an increase in biomass and total number of somatic embryos. For P. mariana a maximum dry weight of 6.3 g1-1 and 3076 embryos m l - 1 occurred in LP medium with 60 mM sucrose after 10-12 days of culture. For P. glauca-engelmannii a maximum dry weight of 4.3 g1-1 and 2278 embryos m l - 1 occurred in LP medium with 60 mM sucrose after 6-8 days culture. For all sucrose concentrations, flesh weight, dry weight and embryo number were closely correlated with packed culture volume and conductivity for P. mariana, and settled culture volume, packed culture volume and conductivity for P. glauca-engelmannii.

Although a variety of non-embryogenic plant cultures have been grown in a range of bioreactor types (for reviews see Scragg and Fowler 1985; Panda et al. 1989; Ten Hoopen et al. 1990), there have been fewer reports of the use of bioreactors for the culture of somatic embryos, for example carrot (Kessell and Carr 1972; Ammirato and Styer 1985), alfalfa (Chen et al. 1987; Stuart et al. 1987), poinsettia (Preil et al. 1988), and sandalwood (Bapat et al. 1990). For conifer species, embryogenic tissue has been used to establish shakeflask suspension cultures (Tautorus et al. 1991) and growth data have been reported (Lulsdorf et al. 1992). The ability to grow embryogenic suspension cultures of conifers on a larger scale is desirable if somatic embryogenesis is to be efficient as a propagation tool. The purpose of this study was to obtain data on growth kinetics for somatic embryos of Picea mariana and the species complex P. glauca-engelmannii when grown separately in a batch bioreactor system, and to determine what culture parameters were highly correlated with culture growth and embryo number. The effect of sucrose concentration on culture growth and embryo number was also investigated, as a test of the reliability of parameters found to be correlated with growth, and because sucrose was completely depleted during growth of shake-flask cultures (Lulsdorf et al. 1992).

Introduction

Materials and methods

Bioreactors provide many advantages for the growth of plant cultures compared to shake-flasks (Ammirato and Styer 1985; Cazzulino et al. 1991). The first advantage is the increased working volume. Second, most bioreactors are designed with either a mechanical or air-lift stirring mechanism, to maintain a nearly homogeneous culture. Third, the cultural and physical environment for the cultures can be controlled for optimum growth (Ammirato and Styer 1985).

Embryogenic cultures. Embryogenic tissue of P. mariana Mill. B.S.P. (black spruce), line BS-D, was initiated from mature zygotic seed as described by Tautorus et al. (1990). Emhryogenic tissue of the species complex P. glauca-engelmannii (interior spruce), line IS-W70, was initiated from immature zygotic seed (Webster et al. 1990), and was obtained from the British Columbia Research Corporation, Vancouver, B.C. Interior spruce is a natural hybrid of P. glauca (Moench) Voss and P. engelmannii Parry, from the interior of British Columbia where their ranges overlap (Owens and Molder 1984). Embryogenic suspension cultures of BS-D and

Correspondence to: D. I. Dunstan

* NRCC No. 33514

47 IS-W70 were prepared using methods previously described (Tautorus et al. 1990). Suspensions were maintained in liquid medium for at least 6 months prior to bioreactor experiments. Suspensions of each species consisted of heterogeneous populations of isodiametric cells, elongated cells, and somatic embryos. Cultures were maintained in 500-ml capacity DeLong flasks containing 200 ml medium after von Arnold and Eriksson (LP, 1981) with 9 ~tM 2,4 dichlorophenoxyacetic acid (2,4-D), 4.4 ~tM N6-benzylaminopurine (BA), and 3 0 m s sucrose. Cultures were agitated on a gyratory shaker (150rpm) under darkness at 24 ---2 ° C. Subcultures were made at 7-day intervals by transferring 40 ml of suspension to fresh medium.

Bioreactor conditions. Two 7.5-1-capacity bioreactors (Microferm MGF-207, New Brunswick Scientific Co., N. J., USA) equipped with galvanic dissolved O2 probes and an O2 analyser were used for batch growth studies. Bioreactor vessels containing 5 1 of LP medium with 9 ~M 2,4-D, 4.4 ~tM BA and either 30, 60, or 90 m s sucrose (pH 5.8) were autoclaved for 30 min at 121 ° C, 104 kPa. After cooling, vessels were inoculated in a laminar flow hood with 200 g (fresh weight) of either 6-day-old BS-D or IS-W70, and 1 1 of conditioned medium (i.e. spent LP medium from shake flasks used for inoculum). Cultures were incubated in the dark and aerated at 0.2 to 0.6 vol. air/vol, medium per minute through a 5-cm-diameter sintered disc sparger. Dissolved 02 was maintained above 80°7o saturation. Agitation rates of 180 to 240 rpm were used for mixing and to prevent settling of cells. Each of the three impellers had six flat blades (1.2 × 0.9 cm) giving a diameter of 5 cm, and were mounted 6 cm, 23 cm, and 34 cm above the bottom of the vessel. Temperature was maintained at 23 + 1° C. All experiments were carried out in duplicate.

Analyses. Three samples (25 ml) were removed from the bioreactots every few days over the course of growth and analysed for fresh weight, dry weight, and number of somatic embryos. In order to find a non-destructive parameter that could be used to predict growth (fresh weight, dry weight) and embryo number, sam-

pies were also analysed for settled culture volume, packed culture volume, pH, osmolarity, and conductivity. Methods of parameter determination were as described by Lulsdorf et al. (1992). Shear effects were evaluated by microscope examination of the samples.

Embryo maturation and germination. For somatic embryo maturation, bioreactor samples containing somatic embryos were inoculated onto LP medium containing 40p~M (---)-abscisic acid (ABA) (A1012, Sigma), 1 [.I.M indole-butyric acid (IBA), 102 mM sucrose, 0.54°7o (w/v) agar (A7002, Sigma) (pH 5.8) following the method of Dunstan et al. (1988, 1991). ABA was prepared as a 1 mM stock solution in acetone/water. Cultures of IS-W70 were incubated in darkness, BS-D was incubated at 3 ~tE m - 2 s -1 for 16 h day from 40-W cool white fluorescent lights. Cultures were incubated at 2 3 + 2 ° C . After approximately 35-55 days, cotyledonary stage embryos (stage 3) were transferred to phytohormonefree half-strength Gina Mohammed-Dunstan (GMD) (Mohammed et al. 1986) agar plates with 15 mM sucrose for germination and plant development.

Statistical analyses. Data were analysed by analysis of variance, mean comparisons were made by protected least significant difference (LSD) at the 5°70 level of probability, and correlation coefficients were calculated using Statgraphics Version 5.0 (Statistical Graphics Corp., Rockville, Md., USA). For calculation of correlation coefficients, repeats were combined after testing the homogeneity of their variances using a Bartlett test (Steel and Torrie 1980).

Results and discussion General observations G r o w t h o f B S - D a n d I S - W 7 0 c u l t u r e s o c c u r r e d u n d e r all b i o r e a c t o r c o n d i t i o n s w i t h n o o b s e r v a b l e signs o f s h e a r stress d u e to m e c h a n i c a l a g i t a t i o n . A g i t a t i o n speeds as

Fig. 1. Somatic embryo of IS-W70 after 6 days culture in a bioreactor containing LP medium (von Arnold and Eriksson 1981) with 60 mM sucrose as described. Bar= 100 ~tm Fig. 2. Cotyledonary stage embryos of BSD after 35 days incubation on LP medium with 40 IXM (-----)abscisic acid (ABA) + 1 ~I.M indole-butyric acid. Cotyledonary stage embryos were derived from somatic embryos grown in a bioreactor for 6 days containing LP medium with 60 mM sucrose as described. Bar = 100 gm Fig. 3. Plantlets of BS-D after 6 weeks incubation on phytohormone-free GMD medium (Mohammed et al. 1986) (following 35 days on ABA medium). Plantlets were derived from somatic embryos grown in a bioreactor containing LP medium with 60 mM sucrose as described. Bar = 1 cm

48 high as 240 rpm produced no visible shear damage. Somatic embryos possessed a dense embryonal mass subtended by a long suspensor system, which was not fractured during culture (Fig. 1). BS-D cultures most commonly grew as dense clusters of somatic embryos, whereas IS-W70 showed a higher frequency of freely suspended individual somatic embryos. Numerous authors have previously reported shearing of plant cells using mechanically-agitated bioreactors, resulting in the recommendation of air-lift bioreactors (see Fowler 1982; Scragg and Fowler 1985; Scragg et al. 1988). However, recent progress has indicated that stirred-tank bioreactors m a y be more conducive for plant cell culture than was at first thought (Scragg et al. 1988). Alfalfa (Chen et al. 1987; Stuart et al. 1987), carrot (Kessel and Carr 1972) and sandalwood (Bapat et al. 1990) somatic embryos have been successfully grown in various mechanically-stirred bioreactors. With BS-D cultures, very little foaming occurred. With IS-W70, the formation of several centimetres of f o a m caused significant accretion of cells above the liquid level on the vessel wall, drive shaft and probes. Further experiments will investigate the use of antifoaming agents with this line. Somatic embryos of BS-D and IS-W70 f r o m bioreactor-grown culture were successfully matured to plantlets following treatment with ABA (Figs. 2, 3). Bioreactor-grown somatic embryos of alfalfa (Chen et al. 1987; Stuart et al. 1987) and sandalwood (Bapat et al. 1990) have also been successfully matured to normal plantlets.

~_

3

"'

2

~:

1

-~-

BS-D

--~-IS-W70

0

2

4

6

8

10

12

14

DAYS Fig. 4. Growth of BS-D and IS-W70 in a bioreactor containing LP medium with 30 mM sucrose. Least significant difference (LSD; p_0.05) values for comparisons of means of BS-D and IS-W70 were 0.43 and 0.31, respectively

days, followed by a stationary phase from day 8. A m a x i m u m dry weight of approximately 4.6 g 1-1 and a specific growth rate of 0.19 d a y - 1 was achieved (Table 1). For comparison, IS-W70 cultures showed no lag phase, a linear phase of approximately 4-8 days, followed by a stationary phase f r o m day 8 (Fig. 4). A m a x i m u m dry weight of 3.5 g1-1 and a specific growth rate of 0.18 day -1 was achieved with IS-W70 (Table 1). Figure 5 shows the production of somatic embryos. Approximately four- and six-fold increases in embryos per millilitre were observed with IS-W70 and BS-D cultures respectively by 8-10 days. The nmnber of somatic embryos decreased thereafter concomitant with a decrease in dry weight. E m b r y o number was closely correlated with dry weight and flesh weight in cultures of each species (Table 2). Yields of somatic embryos for both species in bioreactor culture were approximately 50°7o lower than equivalent cultures grown in shakeflasks (Lulsdorf et al. 1992). During bioreactor culture of alfalfa, decreases in somatic embryo number of 60-80°7o were observed compared to shake-flask cultures (Chen et al. 1987; Stuart et al. 1987). Lower embryo number in bioreactors com-

Growth kinetics and evaluation of parameters Accurate and rapid measurement of cell growth, and assessment of growth kinetics are important for the development and optimization of methods for growth of large-scale plant tissue cultures (Taya et al. 1989; Ryu et al. 1990). Growth kinetics of both species in LP medium with 30 mM sucrose were similar. Changes in total dry weight for BS-D and IS-WT0 cultures are shown in Fig. 4. BS-D cultures showed a lag phase of 1 day, a linear phase of 7

Table 1. Growth characteristics of BS-D and IS:W70 cultured in a bioreactor containing LP medium (von Arnold and Eriksson 1981) with 30, 60 or 90 mM sucrose a Parameters Sucrose concentration Maximum fresh weight (g 1-1) Time to maximum fresh weight (days) Maximum dry weight (g 1-1) Maximum no embryos per ml -I Maximum settled culture volume (ml) Maximum packed culture volume (ml) Lag phase length (days) Linear phase length (days) Specific growth rate, ~ (day-l) b

BS-D 30 mM 102.5 8-10 4.6 2300 10.5 4.2 1 7 0.19

60 mM 127.6 10-12 6.3 3076 15.3 5.5 0 8-10 0.15

IS-W70 90 mM 121.8 10-12 5.2 2772 15.6 5.6 0 10-12 0.13

a Data are the average Of two experiments for each concentration of sucrose b g was calculated over the period of increasing dry weight between day 2 and day 6

30 mM 105.2 8 3.5 2000 9.0 4.5 0 4-8 0.18

60 mM 100.0 10-14 4.3 2278 8.9 4.2 0-1 6-8 0.17

90 mM 95.6 10-14 4.3 1974 7.0 4.3 0-1 10-12 0.12

49 Table 2. Correlation coefficients for BS-D and IS-W70 cultured in a bioreactor containing LP medium with 30 mM sucrose Parameters

BS-D

Fresh weight Dry weight Setted culture volume Packed culture volume Osmolarity Conductivity pH

IS-WT0

Fresh weight

Dry weight

Embryos per ml - ~

--0.76 0.88 - 0.63 - 0.87 0.34 a

0.95 -0.77 0.86 - 0.68 - 0.87 0.40"

0.99 0.96 0.97 0.87 -0.66 -0.83 0.44 a

All correlation coefficients are significant at the 1% level of probability except where indicated

3000

Fresh weight

Dry weight

---

--

Embryos per ml - ~

0.95

0.89 0.96 - 0.78 -0.88 0.35 ~

0.96 0.96 0.96 0.96 - 0.85 -0.85 0.49 ~

0.90 0.91 - 0.74 -0.93 0.32 ~

~ Not significant b S i g n i f i c a n t a t t h e 5 % level

m

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1000

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2

4

6

8

10

12

---*- - I S - W 7 0

14

Fig. 5. Production of somatic embryos of BS-D and IS-W70 in a bioreactor containing LP medium with 30mM sucrose. LSD (p_<0.05) values for comparisons of means of BS-D and IS-W70 were 473.9 and 366, respectively

200

~'150

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<

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-+-=s-w70 0

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o

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DAYS Fi~. 6. Chan~¢s ~n osmo]afity (mOsm) o~ ~ medium wkh 30 m~ sucrose dufin~ biorcactor culture wkh BS-D and IS-WT0. LSD ~ 0 . 0 5 > values ~or comparisons o~ means o~ ~S-D and IS-WT0 were 3.6 and 8.6, respectively

Fig. 7. Changes in conductivity (mS) of LP medium with 30 mM sucrose during bioreactor culture with BS-D and IS-WT0. LSD (p_<0.05) values for comparisons of means of BS-D and IS-W70 were 0.08 and 0.16, respectively

p a r e d to s h a k e - f l a s k s m a y be related to differences in m i x i n g a n d a e r a t i o n . F o r c a r r o t cultures, low c o n c e n t r a tions o f dissolved O2 (i.e. < 16O7o) were f a v o u r a b l e for s o m a t i c e m b r y o f o r m a t i o n (Kessell a n d C a r r 1972). W i t h p o i n s e t t i a s o m a t i c e m b r y o s , dissolved O2 concent r a t i o n s o f less t h a n 60°70 decreased cell division (Preil et al. 1988). A c h i e v e m e n t o f o p t i m a l a e r a t i o n will v a r y with the cell line a n d t y p e o f b i o r e a c t o r used. T h e m a x i m u m settled cell v o l u m e a n d p a c k e d cell v o l u m e for BS-D a n d I S - W 7 0 cultures are s h o w n in Table 1. W i t h BS-D, p a c k e d cell v o l u m e was highly correl a t e d with d r y weight, f l e s h weight a n d e m b r y o n u m b e r , whereas f o r I S - W 7 0 b o t h settled cell v o l u m e a n d p a c k e d cell v o l u m e were highly c o r r e l a t e d with d r y weight, flesh weight a n d e m b r y o n u m b e r in m e d i u m with 30 mM sucrose (Table 2). O s m o l a r i t y initially increased d u r i n g d a y 1, a n d then declined (Fig. 6). D u r i n g the later stages o f g r o w t h (days 10-14), o s m o l a r i t y either r e m a i n e d c o n s t a n t or showed a slight increase c o i n c i d e n t with the s t a t i o n a r y p h a s e a n d cell lysis. O s m o l a r i t y was p o o r l y c o r r e l a t e d with d r y weight, f l e s h weight a n d e m b r y o n u m b e r f o r b o t h species.

50 Changes in the conductivity of the medium for BS-D and IS-W70 cultures are shown in Fig. 7. For each species, conductivity decreased until approximately days 6-8 whereafter it remained constant or a slight increase was observed. Conductivity was negatively correlated with dry weight, fresh weight and embryo number for both species (Table 2). The initial pH after sterilization and inoculation was approximately pH 5.6. Results were similar for each species: within 1 day of culture the pH dropped to pH 4.9 and thereafter slowly increased to 6.5-6.8 at day 14 (data not shown). Changes in medium pH were poorly correlated with dry weight, fresh weight, and embryo number (Table 2), and were not useful for estimation of these growth parameters. However, pH values may be useful for determination of specific growth stages, e.g., pH 6.5-6.8 corresponds with the stationary phase. The parameters (settled culture volume, packed culture volume, conductivity) that could be used to predict dry weight, fresh weight and embryo number in bioreactot cultures were similarly found to be correlatable with shake-flask suspensions of P. mariana and the species complex P. glauca-engelmannii (Lulsdorf et al. 1992). Settled culture volume was recently suggested as a nondestructive parameter for estimation of growth or proliferation of P. sitchensis somatic embryos during shakeflask culture (Krogstrup 1990). However, correlation with growth or embryo number was not reported. Several authors have recommended medium conductivity for evaluating growth kinetics of plant cell suspensions, e.g. Pyrus communis (Ryu et al. 1990), Coffea arabica, Catharanthus roseus, Nicotiana tabacum (Taya et al. 1989), and Glycine m a x (Hahlbrook et al. 1974). In particular, this measurement is non-destructive, rapid and reliable, and can also be used with bioreactors in situ, providing a method for continuous monitoring of growth (Taya et al. 1989; Ryu et al. 1990). It has been suggested that decreases in medium conductivity during plant cell cultivation is due to the uptake of nitrates or combined ionic nutrients (Hahlbrook et al. 1974; Ryu et al. 1990).

Effect o f sucrose concentration

Lulsdorf et al. (1992) suggested that shake-flask suspensions of BS-D and IS-W70 became carbohydrate-limited in medium with 30mM sucrose. Sucrose depletion caused suspension cultures to enter a stationary growth phase, which was characterized by a decline in biomass and by browning of embryos. The effect of sucrose concentration on culture growth and somatic embryo number, was seen when BSD and IS-W70 cultures were inoculated into LP medium with 60 or 90 mM sucrose. With BS-D, the highest dry weight (6.3 g l -~) and fresh weight (127.6 g1-1) were obtained with 60 mM sucrose after 10-12 days culture (Table 1), a specific growth rate of approximately 0.15 day-1. Dry weight and fresh weight of BS-D cultures were significantly different when inoculated into medium with either 60 or 90 mM sucrose compared to

30 mM (p<0.05), suggesting that sucrose was limiting when sucrose was supplied at 30 mM. Although the highest dry weight (4.3 g1-1) of IS-W70 was produced when it was inoculated into medium with 60 or 90 mM sucrose, the effect of sucrose concentration was~not significant. Increased biomass production in both shakeflask (Merillon et al. 1984; Scragg et al. 1990) and bioreactor (Scragg et al. 1990) cultures of C. roseus occurred when sucrose was increased from 60 to 180 mM. However, a decreased growth rate of 0.46 to 0.33 d a y - 1 resuited with the shake-flasks (Scragg et al. 1990). Increased biomass of Dioscorea deltoidea suspension cultures was observed when the sucrose concentration was increased from.15 mM to 45, 90, 135, or 180 mM. Similar growth rates were observed with the different sucrose concentrations (Tal et al. 1982). The number of embryos per millilitre with each species was highest when cultures were inoculated into medium with 60 mM sucrose (Table 1). For IS-W70, the embryo number was significantly higher ( p < 0.05) with 30 and 60 mM sucrose than with 90 m s . However, starting sucrose concentrations had no significant effect on embryo number with BS-D. Packed culture volume and conductivity remained highly correlated with dry weight and fresh weight (>0.91) and embryo number (>0.94) for BS-D cultures inoculated into medium with 60 and 90 mM sucrose. Similarly for IS-W70, settled culture volume, packed culture volume and conductivity were correlated with dry weight and fresh weight ( > 0.83) and embryo number ( > 0.82). This study has demonstrated the potential of a stirred-bioreactor for mass propagation of conifer somatic embryos. Packed culture volume and conductivity were found to be useful non-destructive parameters correlated with fresh weight, dry weight and embryo number. Such parameters could be used to determine the growth status of bioreactor cultures under various experimental or operational conditions. Experiments are in progress to study the effects of embryo maturation from bioreactots in comparison to shake-flasks and solidified medium, as well as the nutrient metabolism during culture. Acknowledgements. We would like to thank Jim Cunningham (Philom Bios Inc., Saskatoon) for valuable advice and assistance during the course of experimentation. This work was funded by the NRC Biotechnology Program, contribution No. CA949-90016 to the British Columbia Research Corp., Vancouver B.C., Petawawa National Forestry Institute, Petawawa, Ont., and the Plant Biotechnology Institute, Saskatoon, Sask.

References

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