Plant Cell Reports
Plant Cell Reports (1996) 15:731 - 736
9 Springer-Verlag1996
Effect of oxygen-enriched aeration on regeneration of rice (Oryza sativa L.) cell culture Akihiro Okamoto 1., Shinobu Kishine ~, Takayasu Hirosawa 1, and Atsuyuki Nakazono 2 1 First Research Center, Nursery Technology Inc., Kitsuregawa-machi, Shioya-gun, Tochigi, 329-14, Japan 2 Third Research Center, Nursery Technology Inc., Kawasaki, Kanagawa 211, Japan * Present address: Nagoya Plant, Kirin Brewery Co. Ltd., Terano, Shinkawa-cho, Nishikasugai-gun, Aichi 452, Japan Received 13 June 1995/Revised version received 25 December 1995 - Communicated by A. Komamine
Abstract The effect of the oxygen concentration in the aeration gas on regeneration from rice cells in bioreactor cultures was investigated. The efficiency of regeneration in cultures aerated with over 40% oxygen was higher than that in a flask culture. In the case of a culture in which the dissolved oxygen(DO) was saturated by aeration with air, the efficiency of regeneration was less than the half that of cultures aerated with 40% oxygen. In cultures with the DO levels controlled at 8,10 and 12 m g / ~ , the efficiency of regeneration was highest at 12 m g / ~ . In the oxygenenriched cultures, although cell aggregation was observed and the color of plantlets was relatively pale, more than 90% of them grew into healthy plants.
K o m a m i n e 1989; T s u k a h a r a and H i r o s a w a 1991; Kobayashi et al. 1992). In one case, 5000 plantlets per liter were obtained ( T a k i g a w a et al. 1992). Data on the regeneration of carrot and alfalfa cells using a bioreactor is also available (Smart et al. 1987; Jay et al. 1992). We have been studying rice r e g e n e r a t i o n for the purpose of supplying large numbers of rice plants, for which it is important to increase the regeneration efficiency in a bioreactor culture. The physical conditions during regeneration; type of bioreactor, temperature of culture, stirring method and speed, concentration of the aeration gases, as well as other parameters, have been investigated. Here, we report on the effect of oxygen concentration on rice regeneration in a bioreactor.
Abbreviations
Material and methods
DO, dissolved oxygen; 2,4-D, 2,4-dichlorophenoxyacetic acid; MES, 2-(N-morpholino) ethanesulfonic acid; rpm, revolution per minute; NAA, 1-naphthaleneacefic acid; vvm, volume per volume per mmnte.
Suspension induction : Mature seeds office (Oryza sativa L. ev. Sasanishiki) were used as explants. Cell culture was initiated as described by Matsuno et al. (1990). Seeds were d e h u s k e d and sterilized with 10% sodium hypochlorite for 30 rain. and then rinsed three times with sterilized water. The sterilized seeds were placed on an induction m e d i u m containing inorganic salts of N6 medium (Chu et aL 1975) supplemented with 10 g/ sucrose, 30 g/~ sorbitol, 12 mM proline, 100 m g / e casein acid hydro]ysate, 4 m g / ~ 2,4-dichlorophen o x y a c e t i c acid (2,4-D), 5 m M 2 - ( N - m o r p h o l i n o ) ethanesulfonic acid (MES) and 2 g/e gellan gum. The pH of the medium was adjusted to 5.8 with KOH. After incubation for 8 days at 25 ~ in the dark, cell dusters formed from the scutellar region of the seeds were removed, transferred onto the same fresh medium and incubated for another 14 days. Then, 1-g portions of fresh cell mass were transferred into 500-ml Erlenmeyer flasks with baffles containing 100 ml of the induction medium without gellan gum. The suspension cultures were subcultured every 7 days. Fresh cells (0.5 g) filtered through of 1000 -/1 m mesh screen and retained on the 100 - , u m mesh screen were inoculated into 100 ml of fresh
Introduction The regeneration of plants from cells has been studied in recent years, and many reports concerning various species have been published. Cells induced from an explant of plants do not differentiated and proliferate in vitro faster than those in organized plant tissues, and the regeneration efficiency from those cells is also much higher than the proliferation efficiency of plant organs. So we can obtain much more clones by regeneration of cells than by tissue culture using the proliferation of plant organs in the same period. For the clonal propagation of a plant in a large scale, the method using regeneration from cells induced from an explant of plants is thus potentially very useful. However, plant regeneration in a liquid medium, in which scale up is thought to be easier than on a solid medium, has been successfully achieved in only a few species. Recently, rice regeneration in liquid cultures has been reported in several studies (Yoshida 1988; Ozawa and Correspondence to: A. Okamoto
732 medium. The flasks were incubated at 25-27 ~ with r o t a t i o n at 80 r e v o l u t i o n per m i n u t e ( r p m ) for a photoperiod of 16 h (at about 2000 lux ). Regeneration culture (flasks) : Regeneration of plantlets f r o m cells was induced in flasks as d e s c r i b e d by Kobayashi et al (1992). Twenty milligrams (fresh weight) of cells retained on the 1000-/1 m mesh screen were inoculated into 100-ml Erlenmeyer flasks containing 20 ml of the regeneration medium, which consisted of N6 salts at half strength, 10 g / e sucrose, 30 g/g sorbitol, 12 m M proline, 2 g / e casein acid hydrolysate, 0.4 rag/g 1naphthaleneacetic acid (NAA), 0.5 m g / g kinetin and 5mM MES. The pH of the medium was adjusted to 5.8 (referred as medium No. 1). The flasks were incubated at 27-30 ~ with rotation at 120 rpm for a photoperiod of 16 h (at about 2000 lux ). After incubation for 4 weeks, the medium in the flasks was completely exchanged with 40 ml fresh medium consisting of N6 salts at half strength, 5 g / g sucrose, 1 5 g / g sorbitol, l g / ~ casein acid hydrolysate, 1 m g / e NAA, 0.5 m g / g kinetin and 2.5mM MES. The pH of the medium was adjusted to 5.8 (referred as medium No.2). The calls were incubated for further 2 weeks and regenerated plantlets that were visible to the naked eye were counted. Regeneration culture (bioreactors) : Glass bioreactors with capacities of 500 ml or 2 e (Shibata Hario Glass, Tokyo, Japan) with working volumes of 250 ml or 1 e , respectively, were used. Each bioreactor was equipped with a water jacket to maintain constant temperature (25 or 30 ~ The bioreactor shape is illustrated in Fig. 1. Air from a compressor and pure oxygen from a bomb were supplied through glass filters at the b o t t o m of the bioreactor. One hundred and twenty-five milligrams (in the case of 500 ml bioreactor) and 500 mg (in the case of the 2 g bioreactor) of cells (fresh weight) retained on the 1000-/1 m mesh screen were respectively inoculated into 125 and 500 ml of medium No. 1. The medium was stirred by an impeller at 40 rpm. The rate of aeration was maintained at 0.1 volume per volume per minute(vvm) for the ~culture period under photoperiod of 16 h (at about 2000 lux ). After incubation for 3 weeks, the medium in
( ) ~
Air
Oxygen Flow meter
Fig.1 Bioreactorshapeand aeration flow. Flow rates of air and oxygenwere controlled by flow controllers and then mixed.
each bioreactor was completely exchanged by 250 ml or 1 g of fresh medium No. 2, and incubation was continued for further 2 weeks. Planflets which were visible to the naked eye were then counted. G r o w t h of o b t a i n e d plantlets : Eighty plantlets r e g e n e r a t e d f r o m cells w e r e p l a n t e d in a m e d i u m containing N6 salts at 1/4 strength and 2 g / e gellan gum in transparent plantboxes (the size were 75 x 75 x 97H ; and 16 plantlets were planted per box). The plantboxes were then placed in a box supplied with air containing 5% carbon dioxide which supported plantlet growth without a carbon source. The planflets were incubated at 30-35 ~ for a photoperiod of 16 h (at about 10,000 lux ). After incubation for 4 weeks, plantlets that survived and those that grew over 2 cm and 10 cm were counted. Measurement of K_ca : The volumetric oxygen transfer coefficient (I~a) was defined as follows. dCL
: K m ( C * - CL )
dt CL : DO concentration of medium (m mol/e ) t : time (hr) d C L / d t : oxygen transfer speed (m m o l / e 9 hr) KL : oxygen transfer coefficient (cm/hr) a : area of the interface between gas and liquid per unit volume (cm2/cm3) C* : DO concentration equilibrated the partial pressure of bubble (m m o l / g ) KLa of the bioreactors were measured using the N2gas sing method. M e a s u r e m e n t of DO c o n c e n t r a t i o n : For the measurement of DO concentration in the flask culture, the same number of flasks for the measurement point was prepared, and at each measurement point the aluminum foil seal was removed and art electric DO probe was installed. The DO concenlration was then measured on a rotary shaker. In the case of the bioreactor culture, the DO concentration was measured by an electric DO probe. Control of oxygen and DO concentration : To control the DO concentration, pure oxygen was mixed with air provided by a compressor, the flow rates of the gases were adjusted by needle valves. The DO concentration was measured by an electric DO probe and was controlled by changing the mixing rates of pure oxygen and air. The DO concentration was checked once or twice per day. Results Effect of oxygen aeration on plantlet regeneration : Fig. 2 shows the r e g e n e r a t i o n efficiencies of plantlets regenerated from cells aerated with air and pure oxygen in the bioreactor at two different temperatures (25 and 30 ~ At both temperatures, much more plantlets were regenerated under aeration with pure oxygen. At 30 ~ 5000 plantlets per ~ medium were regenerated under pure oxygen aeration.
733 6000
" ~ " ' ~ " ~ ' - ' - - ~
=E 5000
E ~9 5000
4000
,~ 4000 3000
--!--
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10000
8000
E 6000 4000
6 z
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o 2
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Fig.2 Regenerationefficiencyof planters from rice cells by aeration with ~r and pure oxygen.(Mediumvolume : 250 ml )
=
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E
r
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'~2000 0
.~
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Fig. 3 Effect of oxygen concentration on the regeneration efficiency of plantlets. The temperatures of cultures were 27-30"C (flask), and 30~ (bioreactor). Medium volume : 40ml (flask), 250 ml (bioreactor) Effect of oxygen concentration on regeneration efficiency : Fig. 3 shows the effect of the oxygen concentration in the aeration gas on the regeneration efficiency of planflets in the bioreactor. The temperature of the culture was at 30 ~ The concentrations of oxygen were 21% (air only), 40, 60 and 80% (mixtures of oxygen and air), and 100% (pure oxygen). The number of plantlets regenerated with air only was 1400 per ~ medium, which was less than that obtained in the flask culture(4800 plantlets). Under aeration with 40, 60, 80 and 100% oxygen, the numbers of
4
6 8 subculture
10 time
12
Fig. ~ Regeneration frequency of rice cells in each subcultured time. Medium volume : 40ml (flask), 250ml(bioreactor) plantlets regenerated per ~ medium ranged from 56008000. Fig. 4 shows regenerated plantlets. Aggregation of cells was observed in the oxygen enriched cultures (over 40%), while the shoots and roots of plantlets grown with oxygen enrichment were much longer and paler green in color than those grown in air or in flask culture. Regeneration frequency in each subcultured time : Fig. 5 shows the r e g e n e r a t i o n f r e q u e n c y o f cells in each subeultured time. F r o m 3 to 10 subculture time, the number of plantlets regenerated in the bioreactor with 100% oxygen was decreased4800 to 1500 per e medium, which was much more than that obtained in the flask culture. On the other hand, few plantlets were obtained in in the bioreaetor with air only. Growth of plantlets regenerated in pure oxygen, air and flask culture : Fig. 6 shows the in vitro survival and growth ratios of planflets on the medium without a carbon source. Plantlets w h i c h s u r v i v e d m e a n s those that remained green and did not die after incubation. More than 90% of plantlets regenerated in pure oxygen grew to over 10 cm compared with 50% in the flask culture, and only 20% in air. Measurement of KLa : Table I shows the KLa values in the bioreactor cultures. These were found to be almost the same under aeration with both air and pure oxygen.
Fig. 4 Regenerated plantlets in cultures with aeration with various concentrations of oxygen. A: Flask (rotation culture; control) B: Air C: Oxygen40% D: Oxygen60% Bar = 1 cm
734 100
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Fig. 6 Growth of plantlets in vitro. Eighty plantlets were planted in 5 plantboxes and incubated at 30-35~ for a photoperiod of 16 h (about 10,000 lux). The ratios of plantlets that survived and those that grew over 2 cm and 10 cm are shown.
Working volume Volume of of bioreactor medium (m[) (ml)
250 250 250 250 1000 1000
125 125 250 250 500 500
1000
1000
1000
1000
Aeration
Speed of stirring (rpm]
air 02 air 02 air 02 air 02
KLa (h-~)
4.7 4.3 6.7
6.0 40 40
5.5 5.5 5.2 4.9
Changes in DO concentration and effects on regeneration under various types of aeration : Fig. 7 shows the DO concentration changes in the flask culture and the cultures under various types of aeration in the bioreactor. Fig. 8 shows the number of plantlets per liter medium in each culture. A c o n s t a n t a e r a t i o n rate (0.1 v v m ) was maintained in "Air(l)". In "Air(2)", the aeration rate was i n c r e a s e d so that DO c o n c e n t r a t i o n indicated the maximum value. In "02 40%", as well as the constant =
~a)
(b) I
i
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o Symbols : o, flask culture 9 ,Air (1)~1 [] ,Air (2)*2 o ,O2 40%"3
10
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Culture time (d)
aeration rate (0.1 vvm), the oxygen concentration in the aeration gas was maintained at 40%. The temperature of the culture was 30 ~ In the flask culture, the DO concentration decreased gradually from 8 to 6 mg/ before the medium exchange, after which it remained at 45 m g / e ; 4700 plantlets per e medium were regenerated. In "Air(l)", the DO concentration decreased from 8 to 4 m g / g in the first 10 days and remained at about 5 mg/ thereafter; 800 plantlets were regenerated. In "Air(2)", the aeration rate increased from 0.1 to 1 vvm by the end of the culture and the DO concentration was maintained 7-8 mg/ throughout; 2300 plantlets were regenerated. In "02 40%", the DO concentration decreased from 19 to 15 mg/ in the first 10 days, remained at about 15 rag/g for another 16 days, and then decreased to 8 rag/e by the end of the culture; 5200 planflets were obtained. Effects of DO concentration in the medium : Fig. 9 shows the effects of DO c o n c e n t r a t i o n on the plantlet regeneration efficiency. The DO concentration was maintained at 8,10 and 12 rag/e in the bioreactor. The
=E
6000
--
5000
,~
4000 3000
1:: ~.
2000
"6 d ;ir
1000 0 Air
Fig.7 Changes of medium DO concentration in flask and bioreactor cultures under various aeration conditions. Arrow(a) and arrow(b) shows the times of medium exchange in the bioreaetor and flask cultures, respectively. *1) The aeration rate was maintained 0.1 vvm during the culture. *2) The aeration rate was increased so that the DO indicated the maximum value. *3) The oxygen concentration was maintained at 40% under a constant aeration rate (0.1 vvm).
Air (1) "1 Air (2)*20240o/o *3
Fig.8 Numbers of plantlets in flask and of bioreactor cultures. The data for flask culture is an average of 5 flasks and each for bioreactor is one data. *1) The aeration rate was maintained at 0.1 vvm during the culture. *2) The aeration rate was increased so that the DO concentration indicated the maximum value. *3) The oxygen concentration was maintained at 40% under a constant aeration rate (0.1 vvm).
Table 1. KLa under various bioreactor culture conditions
r
1000
z
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0
02
20
2000
r
'5
(conbol)
8mg/s
10mg/s
12mg/s
DO concentration Fig. 9 Effect of DO concentration on the regeneration efficiency of plantlets. The aeration rate of cultures, in which the DO concentration was controlled, was maintained 0.1 vvm before medium exchange and 0.05 vvm thereafter. The mixing rate of air and oxygen was changed to control the DO concentration. The aeration rate for "Air" was 0.1vvm.
735
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--~" 3000 2000
l
m Q.
z6
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Air (1)"1 Air (2)*2 0240%TM DO.4 12mg/s
Fig.10 Numbers of plantlets of bioreactor cultures. * 1) The aeration rate was maintained at 0.1 vvm during the culture. *2) The aeration rate was increased so that the DO concentration indicated the maximum value. *3) The oxygen concentration was maintained at 40% under a constant aeration rate (0.1 vvm). *4) The aeration rate of cultures, in which the DO concentration was controlled, was maintained 0.1 vvm before medium exchange and 0.05 vvm thereafter. The mixing rate of air and oxygen was changed to control the DO concentration. Medium volume : 1 ~, (bioreactor)
aeration rate, which controlled the DO concentration, was 0.1 vvm before medium exchange and 0.05 vvm thereafter. The temperature of the culture was 30 ~ The number of planflets r e g e n e r a t e d at 12 mg/ e was 5600 per medium, which was the maximum obtained. At 10 and 8 rag/ ~, 4700 and 2700 plantlets r e s p e c t i v e l y were regenerated, but only 200 was obtained under aeration with air. Fig. 10 shows the result of another experiment which was investigated about the effect on regeneration under two types of aeration and the effect of DO concentration in the medium. Few plantlets were obtained in both types of aeration, of which rate was maintained at 0.1 vvm during the culture and of which rate was increased so that DO concentration indicated the maximum value. The numbers of plantlets regenerated under aeration with 40% oxygen were 3200 per e medium, 4600 at 12 r a g / e of DO concentration. Discussion The efficiency of plantlet regeneration from rice cells was often observed to be very low in bioreactor cultures compared to the flask culture, even when ceils from the same cell line was inoculated. The regeneration efficiency using a bioreactor was found to depend on the condition of culture induction (ex. the period, the place and the person performed the induction), though this was rarely observed in the case of flask cultures. The regeneration of planflets from ceils was thought to be inhibited in bioreactors because of the different physical environment caused, for example, by shear stress due to stirring, aeration and bubbles. We found that the amount of oxygen in the aeration gas was extremely important to increase the
efficiency of regeneration. The regeneration efficiency was increased by using pure oxygen instead of air at both 25 and 30 ~ (Fig. 2). We did not obtain flask culture data in this experiment, but 4000-4800 plantlets per e medium were obtained in the flask culture at 27-30 ~ using the same cell line (data not shown). Differentiation from cells was inhibited in a culture with aeration using only air. A number of studies on aeration or DO conditions in plant cultures have been reported. Carrot required a low dissolved 0 2 tension (16%) for successful somatic embryogenesis (Kessel and Carr 1972) and the number of wheat embryoids was increased at a low O2 level (Carman 1988). In the case of rice, however, the present study shows that a high oxygen concentration had a beneficial effect on the differentiation of cells. On the other hand, the somatic embryo production of carrot under low DO conditions (10%) was inhibited by about 75% (Jay et al 1992), while alfalfa regenerated somatic embryos at a high DO level in a bioreactor (Stuart et al 1987). Vas et al (1992) studied the effect of the m o l e c u l a r o x y g e n a t m o s p h e r e on p r o t o p l a s t culture and o b s e r v e d a significant increase in plant regeneration capacity in the case of rice. Since these studies indicate that the effects of oxygen differ according to species, it has been suggested that more attention should be paid to the role played by oxygen in somatic embryogenesis (Jay et al 1992). The effects of the oxygen concentration in the aeration gas are shown in Fig. 3. A beneficial effect on regeneration was observed at over 40% oxygen, indicating that 40% oxygen is sufficient for the regeneration of rice cells in a bioreactor at the same level as in a flask culture. Cells at the end of every subcultures were inoculated in regeneration cultures for the purpose of verifying the effect of oxygen on regeneration. As shown in Fig 5, much more plantlets were regenerated in cultures with aeration by pure oxygen than those in flasks, and few planflets were obtained in most of cultures with aeration by air. So it can be concluded that planflets were regenerated efficiently by the oxygen-enriched aeration. As shown in Fig. 4, planflets regenerated in oxygenenriched cultures were a paler green than those cultured with air. However, more than 90% of the former grew into healthy plants of over 10 cm in vitro, compared with only 50% of the latter (Fig. 6). It can thus be concluded that no problems arise from using oxygen-enriched air in a rice cell regeneration culture with respect to the growth of the plantlets following the culture. Since the above results indicated that the oxygen transfer speed could be a limiting factor, the KLa values of air and pure oxygen under various bioreactor conditions were measured. However, as shown in Table 1, the KLa values for pure oxygen were almost same as those for air, indicating that the o x y g e n transfer speed was not responsible for the limitation of regeneration in cultures using a bioreactor. Next the DO concentration, a parameter which is related to
736 the concentration of oxygen, was measured. Fig. 7 shows the DO concentration changes in 4 types of cultures, and the numbers of plantlets regenerated in these cultures are shown in Fig. 8. The results suggested that the regeneration efficiency was dependent on the DO level. In the case of Fig.2, much more planflets were regenerated at 30 ~ than 25 ~ although the saturated DO level at 30 ~ may be less than that at 25 ~ The proliferation speed of rice cells at30 ~ was much higher than that at 25 ~ (data not shown), therefore the effect of the culture temperature was also greater than that of the DO in regeneration culture. To verify the effect of DO, control of DO concentration during regeneration was investigated. The regeneration efficiencies when the DO concentration of the medium was controlled at 8, 10 and 12 rag/g are shown in Fig. 9, from which it appears that the regeneration efficiency from rice cells using a bioreactor depends on the DO concentration. On the other hand, an o x y g e n enriched atmosphere had no effect on regeneration in a flask culture (data not shown). A beneficial effect of oxygen-enriched aeration or high concentration of DO on regeneration were also observed in Fig.10. Although each data for bioreactor shown in Fig.2, Fig. 3, Fig.8, Fig.9 and Fig.10 is from a single experiment, we observed evident difference in regeneration of plantlets between cultures with air and oxygen-euriched (or high concentration of DO) cultures from the results of other experiments (data not shown). It can thus be concluded that although regeneration from rice cells in a b i o r e a c t o r culture is inhibited by
environmental parameters, a high DO level contributes to reduce the inhibition. However, we were unable to clarify the mechanism of these effects in the present study. Our future research will be aimed at discovering what parameters inhibit the differentiation office cells and how the DO contributes to this. References Carman JG (1988) Planta 175 : 417-424 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Sci. Sin. 18 : 659- 668 Jay V, Genestier S, Courduroux JC (1992) Hant Cell Rep. 11 : 605-608 Kessel RHJ, Carr AH (1972) J. Exp. Bot. 23 : 996-1007 Kobayashi H, Okii M Hirosawa T (1992) Japan. J. Breed. 42 : 583- 594 Matsuno T, Ishizaki K, Horn ME (1990) Japan. J. Breed. (Suppl 1) 40 : 266-267 Ozawa K, Komamine A (1989) Theor. Appl. Genet. 77 : 205-211 Smart DA, Strickland SG, Walker KA (1987) HortScience 22 : 800-803 Takigawa K, Konishi H, Uchibori H, Sogabe T, Tateyama S, Nakazuno A (1992) Japan. J. Breed. (Suppl. 1) 42 : 18 -19 Tsttkahara M, Itirosawa T (1991) Proc. Ann. Met. Jpn. Soc. Plant. Physiol. : 90 Vaz FB d'Utra, Slamet IH, Khattm A, Cocking EC, Power JB (1992) Plant Cell Rep.ll: 416-418 Yoshida T (1988) Jpn. J. Genet. 63 : 611
Plant Cell Reports (1996) 15:731 - 736
9 Springer-Verlag1996
Effect of oxygen-enriched aeration on regeneration of rice (Oryza sativa L.) cell culture Akihiro Okamoto 1., Shinobu Kishine ~, Takayasu Hirosawa 1, and Atsuyuki Nakazono 2 1 First Research Center, Nursery Technology Inc., Kitsuregawa-machi, Shioya-gun, Tochigi, 329-14, Japan 2 Third Research Center, Nursery Technology Inc., Kawasaki, Kanagawa 211, Japan * Present address: Nagoya Plant, Kirin Brewery Co. Ltd., Terano, Shinkawa-cho, Nishikasugai-gun, Aichi 452, Japan Received 13 June 1995/Revised version received 25 December 1995 - Communicated by A. Komamine
Abstract The effect of the oxygen concentration in the aeration gas on regeneration from rice cells in bioreactor cultures was investigated. The efficiency of regeneration in cultures aerated with over 40% oxygen was higher than that in a flask culture. In the case of a culture in which the dissolved oxygen(DO) was saturated by aeration with air, the efficiency of regeneration was less than the half that of cultures aerated with 40% oxygen. In cultures with the DO levels controlled at 8,10 and 12 m g / ~ , the efficiency of regeneration was highest at 12 m g / ~ . In the oxygenenriched cultures, although cell aggregation was observed and the color of plantlets was relatively pale, more than 90% of them grew into healthy plants.
K o m a m i n e 1989; T s u k a h a r a and H i r o s a w a 1991; Kobayashi et al. 1992). In one case, 5000 plantlets per liter were obtained ( T a k i g a w a et al. 1992). Data on the regeneration of carrot and alfalfa cells using a bioreactor is also available (Smart et al. 1987; Jay et al. 1992). We have been studying rice r e g e n e r a t i o n for the purpose of supplying large numbers of rice plants, for which it is important to increase the regeneration efficiency in a bioreactor culture. The physical conditions during regeneration; type of bioreactor, temperature of culture, stirring method and speed, concentration of the aeration gases, as well as other parameters, have been investigated. Here, we report on the effect of oxygen concentration on rice regeneration in a bioreactor.
Abbreviations
Material and methods
DO, dissolved oxygen; 2,4-D, 2,4-dichlorophenoxyacetic acid; MES, 2-(N-morpholino) ethanesulfonic acid; rpm, revolution per minute; NAA, 1-naphthaleneacefic acid; vvm, volume per volume per mmnte.
Suspension induction : Mature seeds office (Oryza sativa L. ev. Sasanishiki) were used as explants. Cell culture was initiated as described by Matsuno et al. (1990). Seeds were d e h u s k e d and sterilized with 10% sodium hypochlorite for 30 rain. and then rinsed three times with sterilized water. The sterilized seeds were placed on an induction m e d i u m containing inorganic salts of N6 medium (Chu et aL 1975) supplemented with 10 g/ sucrose, 30 g/~ sorbitol, 12 mM proline, 100 m g / e casein acid hydro]ysate, 4 m g / ~ 2,4-dichlorophen o x y a c e t i c acid (2,4-D), 5 m M 2 - ( N - m o r p h o l i n o ) ethanesulfonic acid (MES) and 2 g/e gellan gum. The pH of the medium was adjusted to 5.8 with KOH. After incubation for 8 days at 25 ~ in the dark, cell dusters formed from the scutellar region of the seeds were removed, transferred onto the same fresh medium and incubated for another 14 days. Then, 1-g portions of fresh cell mass were transferred into 500-ml Erlenmeyer flasks with baffles containing 100 ml of the induction medium without gellan gum. The suspension cultures were subcultured every 7 days. Fresh cells (0.5 g) filtered through of 1000 -/1 m mesh screen and retained on the 100 - , u m mesh screen were inoculated into 100 ml of fresh
Introduction The regeneration of plants from cells has been studied in recent years, and many reports concerning various species have been published. Cells induced from an explant of plants do not differentiated and proliferate in vitro faster than those in organized plant tissues, and the regeneration efficiency from those cells is also much higher than the proliferation efficiency of plant organs. So we can obtain much more clones by regeneration of cells than by tissue culture using the proliferation of plant organs in the same period. For the clonal propagation of a plant in a large scale, the method using regeneration from cells induced from an explant of plants is thus potentially very useful. However, plant regeneration in a liquid medium, in which scale up is thought to be easier than on a solid medium, has been successfully achieved in only a few species. Recently, rice regeneration in liquid cultures has been reported in several studies (Yoshida 1988; Ozawa and Correspondence to: A. Okamoto
732 medium. The flasks were incubated at 25-27 ~ with r o t a t i o n at 80 r e v o l u t i o n per m i n u t e ( r p m ) for a photoperiod of 16 h (at about 2000 lux ). Regeneration culture (flasks) : Regeneration of plantlets f r o m cells was induced in flasks as d e s c r i b e d by Kobayashi et al (1992). Twenty milligrams (fresh weight) of cells retained on the 1000-/1 m mesh screen were inoculated into 100-ml Erlenmeyer flasks containing 20 ml of the regeneration medium, which consisted of N6 salts at half strength, 10 g / e sucrose, 30 g/g sorbitol, 12 m M proline, 2 g / e casein acid hydrolysate, 0.4 rag/g 1naphthaleneacetic acid (NAA), 0.5 m g / g kinetin and 5mM MES. The pH of the medium was adjusted to 5.8 (referred as medium No. 1). The flasks were incubated at 27-30 ~ with rotation at 120 rpm for a photoperiod of 16 h (at about 2000 lux ). After incubation for 4 weeks, the medium in the flasks was completely exchanged with 40 ml fresh medium consisting of N6 salts at half strength, 5 g / g sucrose, 1 5 g / g sorbitol, l g / ~ casein acid hydrolysate, 1 m g / e NAA, 0.5 m g / g kinetin and 2.5mM MES. The pH of the medium was adjusted to 5.8 (referred as medium No.2). The calls were incubated for further 2 weeks and regenerated plantlets that were visible to the naked eye were counted. Regeneration culture (bioreactors) : Glass bioreactors with capacities of 500 ml or 2 e (Shibata Hario Glass, Tokyo, Japan) with working volumes of 250 ml or 1 e , respectively, were used. Each bioreactor was equipped with a water jacket to maintain constant temperature (25 or 30 ~ The bioreactor shape is illustrated in Fig. 1. Air from a compressor and pure oxygen from a bomb were supplied through glass filters at the b o t t o m of the bioreactor. One hundred and twenty-five milligrams (in the case of 500 ml bioreactor) and 500 mg (in the case of the 2 g bioreactor) of cells (fresh weight) retained on the 1000-/1 m mesh screen were respectively inoculated into 125 and 500 ml of medium No. 1. The medium was stirred by an impeller at 40 rpm. The rate of aeration was maintained at 0.1 volume per volume per minute(vvm) for the ~culture period under photoperiod of 16 h (at about 2000 lux ). After incubation for 3 weeks, the medium in
( ) ~
Air
Oxygen Flow meter
Fig.1 Bioreactorshapeand aeration flow. Flow rates of air and oxygenwere controlled by flow controllers and then mixed.
each bioreactor was completely exchanged by 250 ml or 1 g of fresh medium No. 2, and incubation was continued for further 2 weeks. Planflets which were visible to the naked eye were then counted. G r o w t h of o b t a i n e d plantlets : Eighty plantlets r e g e n e r a t e d f r o m cells w e r e p l a n t e d in a m e d i u m containing N6 salts at 1/4 strength and 2 g / e gellan gum in transparent plantboxes (the size were 75 x 75 x 97H ; and 16 plantlets were planted per box). The plantboxes were then placed in a box supplied with air containing 5% carbon dioxide which supported plantlet growth without a carbon source. The planflets were incubated at 30-35 ~ for a photoperiod of 16 h (at about 10,000 lux ). After incubation for 4 weeks, plantlets that survived and those that grew over 2 cm and 10 cm were counted. Measurement of K_ca : The volumetric oxygen transfer coefficient (I~a) was defined as follows. dCL
: K m ( C * - CL )
dt CL : DO concentration of medium (m mol/e ) t : time (hr) d C L / d t : oxygen transfer speed (m m o l / e 9 hr) KL : oxygen transfer coefficient (cm/hr) a : area of the interface between gas and liquid per unit volume (cm2/cm3) C* : DO concentration equilibrated the partial pressure of bubble (m m o l / g ) KLa of the bioreactors were measured using the N2gas sing method. M e a s u r e m e n t of DO c o n c e n t r a t i o n : For the measurement of DO concentration in the flask culture, the same number of flasks for the measurement point was prepared, and at each measurement point the aluminum foil seal was removed and art electric DO probe was installed. The DO concenlration was then measured on a rotary shaker. In the case of the bioreactor culture, the DO concentration was measured by an electric DO probe. Control of oxygen and DO concentration : To control the DO concentration, pure oxygen was mixed with air provided by a compressor, the flow rates of the gases were adjusted by needle valves. The DO concentration was measured by an electric DO probe and was controlled by changing the mixing rates of pure oxygen and air. The DO concentration was checked once or twice per day. Results Effect of oxygen aeration on plantlet regeneration : Fig. 2 shows the r e g e n e r a t i o n efficiencies of plantlets regenerated from cells aerated with air and pure oxygen in the bioreactor at two different temperatures (25 and 30 ~ At both temperatures, much more plantlets were regenerated under aeration with pure oxygen. At 30 ~ 5000 plantlets per ~ medium were regenerated under pure oxygen aeration.
733 6000
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Fig.2 Regenerationefficiencyof planters from rice cells by aeration with ~r and pure oxygen.(Mediumvolume : 250 ml )
=
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Fig. 3 Effect of oxygen concentration on the regeneration efficiency of plantlets. The temperatures of cultures were 27-30"C (flask), and 30~ (bioreactor). Medium volume : 40ml (flask), 250 ml (bioreactor) Effect of oxygen concentration on regeneration efficiency : Fig. 3 shows the effect of the oxygen concentration in the aeration gas on the regeneration efficiency of planflets in the bioreactor. The temperature of the culture was at 30 ~ The concentrations of oxygen were 21% (air only), 40, 60 and 80% (mixtures of oxygen and air), and 100% (pure oxygen). The number of plantlets regenerated with air only was 1400 per ~ medium, which was less than that obtained in the flask culture(4800 plantlets). Under aeration with 40, 60, 80 and 100% oxygen, the numbers of
4
6 8 subculture
10 time
12
Fig. ~ Regeneration frequency of rice cells in each subcultured time. Medium volume : 40ml (flask), 250ml(bioreactor) plantlets regenerated per ~ medium ranged from 56008000. Fig. 4 shows regenerated plantlets. Aggregation of cells was observed in the oxygen enriched cultures (over 40%), while the shoots and roots of plantlets grown with oxygen enrichment were much longer and paler green in color than those grown in air or in flask culture. Regeneration frequency in each subcultured time : Fig. 5 shows the r e g e n e r a t i o n f r e q u e n c y o f cells in each subeultured time. F r o m 3 to 10 subculture time, the number of plantlets regenerated in the bioreactor with 100% oxygen was decreased4800 to 1500 per e medium, which was much more than that obtained in the flask culture. On the other hand, few plantlets were obtained in in the bioreaetor with air only. Growth of plantlets regenerated in pure oxygen, air and flask culture : Fig. 6 shows the in vitro survival and growth ratios of planflets on the medium without a carbon source. Plantlets w h i c h s u r v i v e d m e a n s those that remained green and did not die after incubation. More than 90% of plantlets regenerated in pure oxygen grew to over 10 cm compared with 50% in the flask culture, and only 20% in air. Measurement of KLa : Table I shows the KLa values in the bioreactor cultures. These were found to be almost the same under aeration with both air and pure oxygen.
Fig. 4 Regenerated plantlets in cultures with aeration with various concentrations of oxygen. A: Flask (rotation culture; control) B: Air C: Oxygen40% D: Oxygen60% Bar = 1 cm
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Fig. 6 Growth of plantlets in vitro. Eighty plantlets were planted in 5 plantboxes and incubated at 30-35~ for a photoperiod of 16 h (about 10,000 lux). The ratios of plantlets that survived and those that grew over 2 cm and 10 cm are shown.
Working volume Volume of of bioreactor medium (m[) (ml)
250 250 250 250 1000 1000
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1000
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Aeration
Speed of stirring (rpm]
air 02 air 02 air 02 air 02
KLa (h-~)
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Changes in DO concentration and effects on regeneration under various types of aeration : Fig. 7 shows the DO concentration changes in the flask culture and the cultures under various types of aeration in the bioreactor. Fig. 8 shows the number of plantlets per liter medium in each culture. A c o n s t a n t a e r a t i o n rate (0.1 v v m ) was maintained in "Air(l)". In "Air(2)", the aeration rate was i n c r e a s e d so that DO c o n c e n t r a t i o n indicated the maximum value. In "02 40%", as well as the constant =
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aeration rate (0.1 vvm), the oxygen concentration in the aeration gas was maintained at 40%. The temperature of the culture was 30 ~ In the flask culture, the DO concentration decreased gradually from 8 to 6 mg/ before the medium exchange, after which it remained at 45 m g / e ; 4700 plantlets per e medium were regenerated. In "Air(l)", the DO concentration decreased from 8 to 4 m g / g in the first 10 days and remained at about 5 mg/ thereafter; 800 plantlets were regenerated. In "Air(2)", the aeration rate increased from 0.1 to 1 vvm by the end of the culture and the DO concentration was maintained 7-8 mg/ throughout; 2300 plantlets were regenerated. In "02 40%", the DO concentration decreased from 19 to 15 mg/ in the first 10 days, remained at about 15 rag/g for another 16 days, and then decreased to 8 rag/e by the end of the culture; 5200 planflets were obtained. Effects of DO concentration in the medium : Fig. 9 shows the effects of DO c o n c e n t r a t i o n on the plantlet regeneration efficiency. The DO concentration was maintained at 8,10 and 12 rag/e in the bioreactor. The
=E
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--
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,~
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2000
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Fig.7 Changes of medium DO concentration in flask and bioreactor cultures under various aeration conditions. Arrow(a) and arrow(b) shows the times of medium exchange in the bioreaetor and flask cultures, respectively. *1) The aeration rate was maintained 0.1 vvm during the culture. *2) The aeration rate was increased so that the DO indicated the maximum value. *3) The oxygen concentration was maintained at 40% under a constant aeration rate (0.1 vvm).
Air (1) "1 Air (2)*20240o/o *3
Fig.8 Numbers of plantlets in flask and of bioreactor cultures. The data for flask culture is an average of 5 flasks and each for bioreactor is one data. *1) The aeration rate was maintained at 0.1 vvm during the culture. *2) The aeration rate was increased so that the DO concentration indicated the maximum value. *3) The oxygen concentration was maintained at 40% under a constant aeration rate (0.1 vvm).
Table 1. KLa under various bioreactor culture conditions
r
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20
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r
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10mg/s
12mg/s
DO concentration Fig. 9 Effect of DO concentration on the regeneration efficiency of plantlets. The aeration rate of cultures, in which the DO concentration was controlled, was maintained 0.1 vvm before medium exchange and 0.05 vvm thereafter. The mixing rate of air and oxygen was changed to control the DO concentration. The aeration rate for "Air" was 0.1vvm.
735
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Fig.10 Numbers of plantlets of bioreactor cultures. * 1) The aeration rate was maintained at 0.1 vvm during the culture. *2) The aeration rate was increased so that the DO concentration indicated the maximum value. *3) The oxygen concentration was maintained at 40% under a constant aeration rate (0.1 vvm). *4) The aeration rate of cultures, in which the DO concentration was controlled, was maintained 0.1 vvm before medium exchange and 0.05 vvm thereafter. The mixing rate of air and oxygen was changed to control the DO concentration. Medium volume : 1 ~, (bioreactor)
aeration rate, which controlled the DO concentration, was 0.1 vvm before medium exchange and 0.05 vvm thereafter. The temperature of the culture was 30 ~ The number of planflets r e g e n e r a t e d at 12 mg/ e was 5600 per medium, which was the maximum obtained. At 10 and 8 rag/ ~, 4700 and 2700 plantlets r e s p e c t i v e l y were regenerated, but only 200 was obtained under aeration with air. Fig. 10 shows the result of another experiment which was investigated about the effect on regeneration under two types of aeration and the effect of DO concentration in the medium. Few plantlets were obtained in both types of aeration, of which rate was maintained at 0.1 vvm during the culture and of which rate was increased so that DO concentration indicated the maximum value. The numbers of plantlets regenerated under aeration with 40% oxygen were 3200 per e medium, 4600 at 12 r a g / e of DO concentration. Discussion The efficiency of plantlet regeneration from rice cells was often observed to be very low in bioreactor cultures compared to the flask culture, even when ceils from the same cell line was inoculated. The regeneration efficiency using a bioreactor was found to depend on the condition of culture induction (ex. the period, the place and the person performed the induction), though this was rarely observed in the case of flask cultures. The regeneration of planflets from ceils was thought to be inhibited in bioreactors because of the different physical environment caused, for example, by shear stress due to stirring, aeration and bubbles. We found that the amount of oxygen in the aeration gas was extremely important to increase the
efficiency of regeneration. The regeneration efficiency was increased by using pure oxygen instead of air at both 25 and 30 ~ (Fig. 2). We did not obtain flask culture data in this experiment, but 4000-4800 plantlets per e medium were obtained in the flask culture at 27-30 ~ using the same cell line (data not shown). Differentiation from cells was inhibited in a culture with aeration using only air. A number of studies on aeration or DO conditions in plant cultures have been reported. Carrot required a low dissolved 0 2 tension (16%) for successful somatic embryogenesis (Kessel and Carr 1972) and the number of wheat embryoids was increased at a low O2 level (Carman 1988). In the case of rice, however, the present study shows that a high oxygen concentration had a beneficial effect on the differentiation of cells. On the other hand, the somatic embryo production of carrot under low DO conditions (10%) was inhibited by about 75% (Jay et al 1992), while alfalfa regenerated somatic embryos at a high DO level in a bioreactor (Stuart et al 1987). Vas et al (1992) studied the effect of the m o l e c u l a r o x y g e n a t m o s p h e r e on p r o t o p l a s t culture and o b s e r v e d a significant increase in plant regeneration capacity in the case of rice. Since these studies indicate that the effects of oxygen differ according to species, it has been suggested that more attention should be paid to the role played by oxygen in somatic embryogenesis (Jay et al 1992). The effects of the oxygen concentration in the aeration gas are shown in Fig. 3. A beneficial effect on regeneration was observed at over 40% oxygen, indicating that 40% oxygen is sufficient for the regeneration of rice cells in a bioreactor at the same level as in a flask culture. Cells at the end of every subcultures were inoculated in regeneration cultures for the purpose of verifying the effect of oxygen on regeneration. As shown in Fig 5, much more plantlets were regenerated in cultures with aeration by pure oxygen than those in flasks, and few planflets were obtained in most of cultures with aeration by air. So it can be concluded that planflets were regenerated efficiently by the oxygen-enriched aeration. As shown in Fig. 4, planflets regenerated in oxygenenriched cultures were a paler green than those cultured with air. However, more than 90% of the former grew into healthy plants of over 10 cm in vitro, compared with only 50% of the latter (Fig. 6). It can thus be concluded that no problems arise from using oxygen-enriched air in a rice cell regeneration culture with respect to the growth of the plantlets following the culture. Since the above results indicated that the oxygen transfer speed could be a limiting factor, the KLa values of air and pure oxygen under various bioreactor conditions were measured. However, as shown in Table 1, the KLa values for pure oxygen were almost same as those for air, indicating that the o x y g e n transfer speed was not responsible for the limitation of regeneration in cultures using a bioreactor. Next the DO concentration, a parameter which is related to
736 the concentration of oxygen, was measured. Fig. 7 shows the DO concentration changes in 4 types of cultures, and the numbers of plantlets regenerated in these cultures are shown in Fig. 8. The results suggested that the regeneration efficiency was dependent on the DO level. In the case of Fig.2, much more planflets were regenerated at 30 ~ than 25 ~ although the saturated DO level at 30 ~ may be less than that at 25 ~ The proliferation speed of rice cells at30 ~ was much higher than that at 25 ~ (data not shown), therefore the effect of the culture temperature was also greater than that of the DO in regeneration culture. To verify the effect of DO, control of DO concentration during regeneration was investigated. The regeneration efficiencies when the DO concentration of the medium was controlled at 8, 10 and 12 rag/g are shown in Fig. 9, from which it appears that the regeneration efficiency from rice cells using a bioreactor depends on the DO concentration. On the other hand, an o x y g e n enriched atmosphere had no effect on regeneration in a flask culture (data not shown). A beneficial effect of oxygen-enriched aeration or high concentration of DO on regeneration were also observed in Fig.10. Although each data for bioreactor shown in Fig.2, Fig. 3, Fig.8, Fig.9 and Fig.10 is from a single experiment, we observed evident difference in regeneration of plantlets between cultures with air and oxygen-euriched (or high concentration of DO) cultures from the results of other experiments (data not shown). It can thus be concluded that although regeneration from rice cells in a b i o r e a c t o r culture is inhibited by
environmental parameters, a high DO level contributes to reduce the inhibition. However, we were unable to clarify the mechanism of these effects in the present study. Our future research will be aimed at discovering what parameters inhibit the differentiation office cells and how the DO contributes to this. References Carman JG (1988) Planta 175 : 417-424 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Sci. Sin. 18 : 659- 668 Jay V, Genestier S, Courduroux JC (1992) Hant Cell Rep. 11 : 605-608 Kessel RHJ, Carr AH (1972) J. Exp. Bot. 23 : 996-1007 Kobayashi H, Okii M Hirosawa T (1992) Japan. J. Breed. 42 : 583- 594 Matsuno T, Ishizaki K, Horn ME (1990) Japan. J. Breed. (Suppl 1) 40 : 266-267 Ozawa K, Komamine A (1989) Theor. Appl. Genet. 77 : 205-211 Smart DA, Strickland SG, Walker KA (1987) HortScience 22 : 800-803 Takigawa K, Konishi H, Uchibori H, Sogabe T, Tateyama S, Nakazuno A (1992) Japan. J. Breed. (Suppl. 1) 42 : 18 -19 Tsttkahara M, Itirosawa T (1991) Proc. Ann. Met. Jpn. Soc. Plant. Physiol. : 90 Vaz FB d'Utra, Slamet IH, Khattm A, Cocking EC, Power JB (1992) Plant Cell Rep.ll: 416-418 Yoshida T (1988) Jpn. J. Genet. 63 : 611
